Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes
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《核酸研究医学期刊》
Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway and 1 Institut National de la Santé et de la Recherche Médicale Unité 429, H?pital Necker-Enfants Malades, 75015 Paris, France
* To whom correspondence should be addressed. Tel: +47 73598695; Fax: +47 73598801; Email: hans.krokan@medisin.ntnu.no
ABSTRACT
Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POL?, POL, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POL?, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POL? dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).
INTRODUCTION
Uracil in DNA occurs as a result of deamination of cytosine and incorporation of dUMP during replication. Deamination of cytosine occurs at a rate of 100–500 per human cell per day, yielding mutagenic U/G mispairs which, unless repaired, result in GC to AT transitions upon replication (1). Incorporation of dUMP during replication results in U/A pairs which are not miscoding, but which may yield cytotoxic and potentially mutagenic abasic (AP) sites (2). Uracil in DNA may also affect transcriptional fidelity (3), as well as binding of transcription factors (4). A recently identified source of uracil in the genome is the enzymatic deamination of cytosine to uracil by activation-induced cytidine deaminase (AID) in the process of somatic hypermutation and antibody class switch in B-cells (5). Uracil is recognized by a uracil-DNA glycosylase (UDG) activity, which cleaves the N-glycosylic bond leaving an AP-site in DNA. Human cells contain at least four types of UDG; mitochondrial UNG1 and nuclear UNG2, SMUG1, TDG and MBD4, which have overlapping substrate specificities (6). Their specific functions are still unclear. Among these glycosylases, UNG proteins are by far the catalytically most efficient (6,7). UNG1 and nuclear UNG2 are both encoded by the UNG-gene and have a common catalytic domain, but different N-terminal sequences required for subcellular sorting (8). Upon mitochondrial import the preform of UNG1 is processed to a mature form lacking 29 N-terminal amino acid residues (9). AP-sites in nuclear DNA are repaired by either single-nucleotide (short-patch) base excision repair (BER) or via replacement of several nucleotides (long-patch BER). Short-patch BER requires an AP endonuclease, POL? and DNA ligase III or possibly also DNA ligase I, while long-patch BER depends on flap endonuclease I (FEN-1), and may require proliferating cell nuclear antigen (PCNA), DNA polymerases / and DNA ligase I. POL? has also been suggested to be required for long-patch repair either for insertion of the first nucleotide (10) or for strand displacement (11). An APE1-independent short-patch BER pathway has recently been suggested. Thus, the bifunctional DNA glycosylases NEIL1 and NEIL2 carry out ?, -elimination after excision of damaged bases generating a one-nucleotide gap flanked by 3'-phosphate and 5'-phosphate termini. The 3'-phosphate terminus may subsequently be converted to a 3'-OH terminus by polynucleotide kinase (PNK), thus setting up for short-patch repair (12).
In DNA repair and related processes, there is evidence for the existence of functional multi-protein complexes. Thus, multi-protein complexes involved in eukaryote transcription and replication have been reported (13,14). A group of proteins that associates with DNA repair protein BRCA1 has been identified and named BRCA1-associated genome surveillance complex (BASC). It includes DNA repair proteins MSH2, MSH6, MLH1, ATM, BLM and the RAD50–MRE11–NBS1 protein complex, PCNA and RF-C (15). Furthermore, a multi-protein complex that connects Fanconi anemia and Bloom syndrome has recently been reported (16). Many interactions between the proteins in the initial step of nucleotide excision repair (NER) have also been reported and the existence of a multiprotein repairosome complex was suggested (17), although a later study did not find evidence in support of such a complex (18). Nevertheless, it is evident that many interactions do occur and it may seem likely that the mechanism of NER at least in part relies on formation of complexes (19). There is also evidence for complex formation in BER. Thus, a 180 kDa protein complex that repairs uracil-containing DNA was isolated from bovine testis using DNA polymerase ?-affinity chromatography (20). Direct molecular interactions between UNG2 and other BER factors, e.g. RPA (21) and PCNA (22) have also been reported. However, PCNA interacts with multiple partners, e.g. POL, POL, RF-C, DNA ligase I, FEN-1, POL?, APE1 and XRCC1, all of which are involved in long-patch BER, and all, except POL? and XRCC1, in DNA replication (23–25). Most likely PCNA engages in different complexes, but it is also possible that the three binding sites in this homotrimeric protein bind different proteins. POL? also interacts directly with ligase I (26), XRCC1 (27) and APE1 (28). In addition, XRCC1 interacts with DNA ligase III (27,29) with poly(ADP-ribose) polymerase (PARP) through one of its BRCT-modules (30), PNK (31), APE1 (32) and PCNA (24) acting as a scaffold protein in BER and single-strand break repair (33).
Given the plethora of interactions between BER proteins, it seemed likely that it would be possible to isolate a complex for nuclear BER of uracil-containing DNA, using a UNG2-specific antibody or other BER-antibodies. The present work demonstrates the presence and properties of an UNG2-containing protein complex proficient in complete repair of uracil-containing DNA.
MATERIALS AND METHODS
Synthetic oligonucleotides were from MedProbe (Oslo, Norway). dTTP and dCTP (3000 Ci/mmol) were from Amersham Biosciences. Primary antibodies against POL (mouse monoclonal) were from Transduction Laboratories, Lexington, KY, antibodies against PCNA (ab29, PC10), POL? (mouse monoclonal) and XRCC1 (rabbit polyclonal) were from Abcam Ltd, UK, and antibodies against APE1 (rabbit polyclonal) from Novus Biologicals Inc., Littleton, CO, USA. Rabbit antiserum to APE2 was kindly provided by Magnar Bj?r?s (Centre of Molecular Biology and Department of Molecular Biology, Oslo). Human recombinant POL? and neutralizing antibodies to POL? were generous gifts from Dr S. H. Wilson (Laboratory of Structural Biology, NIH). Paramagnetic Protein A-beads were from Dynal, Oslo, Norway. A neutralizing polyclonal rabbit anti-hSMUG1 IgG (PSM1) was prepared as described previously (7). Silver-staining of protein gels was carried out using the ProteosilverTM Plus, Silver Stain Kit (Sigma-Aldrich Co.).
DNA substrates
Covalently closed circular DNA (cccDNA) substrates were prepared essentially as described previously (34). Briefly, 20 μg of ssDNA were annealed to 4.2 μg of a 5' phosphorylated 22mer complementary oligonucleotide containing either a uracil or normal base (molar ratio 1:30) (Figure 1A). Synthesis of duplex DNA was carried out in the presence of T4 DNA polymerase, T4 DNA ligase and T4 gene 32 ssDNA-binding protein at 37°C for 2 h. cccDNA duplex molecules were purified by CsCl gradient centrifugation. The purity of the substrate prepared was estimated by agarose gel electrophoresis (Figure 1B). Only DNA substrate containing >95% cccDNA was used in BER assays. By site-directed mutagenesis of a pGEM-3zf(+) Phagemide, we prepared a G to A mutation to be able to prepare substrates containing U/A and U/G in the same sequence context. For AP-site substrate, uracil-containing cccDNA was incubated with purified catalytic domain of UNG (35) just before use. Complete removal of uracil was confirmed by further incubation of an aliquot of this DNA with purified APE1 protein resulting in complete conversion of form I (supercoiled closed circular DNA) to form II (nicked circular DNA) detected by agarose gel electrophoresis (data not shown). For generation of AP-nick substrate DNA, uracil-containing plasmid was incubated with purified catalytic domain of UNG and purified APE1 protein.
Figure 1. Substrate and strategy for BER studies. (A) As substrate for the BER assay we used double-stranded covalently closed circular pGEM-3Zf(+) with U/G, U/A, AP-site/G or nicked AP-site/A in the same position. The cleavage sites for restriction enzymes used for mapping of the repair patches are indicated. The shaded region corresponds to the repaired patch. (B) The quality of the substrate in terms of the amount of covalently closed circular plasmid DNA (form I) and nicked circular forms (form II) purified by CsCl/ethidium bromide equilibrium centrifugation. Purity was assessed by running 100 ng of DNA on 1% agarose gel with PstI digested DNA as marker. (C) Specificity of anti-UNG-antibodies PU101 and PU1sub. Recombinant UNG2, the mitochondrial form UNG129 and the catalytic UNG-domain were separated by polyacrylamide gel electrophoresis and subjected to western analysis using either PU101 or PU1sub, as indicated. (D) Structure of the preform of UNG1, UNG129, UNG2 and the catalytic domain UNG84.
UNG antibodies
Neutralizing anti-UNG-antibody PU101 against the catalytic domain has been described previously (35). Polyclonal anti-UNG2 PU1 was prepared following the same procedure. PU1sub antibody against the N-terminal region of the human UNG2 was prepared by passing PU1 IgG over a matrix containing the recombinant C-terminal catalytic UNG-domain bound to NHS-activated Sepharose (Amersham Biosciences). The IgGs in the flow-through proved to be specific for the UNG2 N-terminal regulatory domain as judged from western analysis (Figure 1C and D), and did not inhibit UNG enzyme activity.
HeLa Tet-On cells (HTO) overexpressing UNG2 (HTO-UNG2)
UNG2 cDNA was cloned into the EcoRI/XbaI sites of vector pTRE and the construct (pTRE-UNG2) co-transfected with pTK-Hyg into HTO cells. Hygromycin resistant clones were selected and subcloned by dilution. The subclone that repeatedly gave the best expression after induction, HTO-UNG2-45, was used in the present study.
Culture of cell lines and preparation of whole cell extracts
HaCaT, HeLa S3 and HTO-UNG2 cells were cultured in DMEM with 10% fetal calf serum (FCS), 0.03% glutamine and 0.1 mg/ml gentamicin at 5% CO2. Human myeloma cell line JJN-3 was cultured under similar conditions but in RPMI 1640 medium. Peripheral blood lymphocytes were obtained by density gradient centrifugation of buffy coat over LymphoprepTM (Nycomed, Norway). The UNG–/– lymphoblastoid cell line was from patient 2 (36) and carried a Phe251Ser homozygous mutation. Cells were grown in RPMI 1640, with 0.03% glutamine, 10% heat-inactivated FCS, and 100 U/ml penicillin and 100 μg/ml streptomycin at 5% CO2.
Whole cell extracts were prepared essentially as described by Tanaka et al. (37). Briefly, cells were pelleted at 215 g and resuspended at 1x packed cell volume in buffer I and 1x packed cell volume of buffer II . The mixture was rocked at 4°C for 2 h and cell debris was pelleted at 22 000 g at 4°C for 10 min. The supernatant was recovered and protein concentration measured using the Bio-Rad protein assay. Extracts were snap frozen in liquid nitrogen and stored in small aliquots at –80°C.
Preparation of BER complex UNG2-ARC
PU1sub IgGs were covalently linked to magnetic Dynabeads? Protein A using dimethyl pimelimidate dihydrochloride (DMP) according to instructions from the manufacturer (Dynal, Norway) with minor modifications: 400 μg protein from whole HeLa cell extract was mixed with 5 μl of the antibody-coated beads or otherwise indicated, and kept in suspension under constant and gentle rocking for 4 h at 4°C. The beads were washed three times with 10 mM Tris–HCl, pH 7.5, transferred to a new tube, washed once more in the same buffer and resuspended in appropriate buffer for further use. For control experiments, we prepared beads linked to the same amount of pre-immune IgG from the same rabbit (pre-immune-IgG), and non-immunized rabbit serum (non-immune-IgG).
BER assay
The BER mixtures (50 μl) contained (final) 40 mM HEPES–KOH (pH 7.8), 70 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 2 mM ATP, 20 μM dATP, 20 μM dGTP, 8 μM dCTP or dTTP depending on the isotope used, 4.4 mM phosphocreatine, 2.5 μg creatine kinase, 18 μg BSA, 2μCi dCTP or dTTP, 50 μg whole cell extract (measured as protein) or UNG2-ARC (5 μl beads) or otherwise immunoprecipitated material, and 300 ng cccDNA substrate if not otherwise indicated. For BER assay experiments, the beads were resuspended in 10 mM Tris–HCl, pH 8.0 containing Complete? protease inhibitor and 7% glycerol (final). The repair mixtures were incubated at 30°C for the indicated times (usually 60 min), and stopped by adding (final) 20 mM EDTA and 80 ng/μl RNase A (37°C, 10 min), and further incubated with (final) 190 ng/μl proteinase K and 0.5% SDS (37°C, 30 min). The repair products were purified by phenol/chloroform extraction and ethanol/salt precipitation. DNA was digested with indicated restriction enzymes (Figure 1), and analyzed by electrophoresis in 12% denaturing polyacrylamide gels and phosphorimaging (Fuji, BAS-1800II) of the dried gels.
Western analysis
Proteins were separated on pre-cast 10% denaturing NuPAGE gels (InvitrogenTM, Life Technologies) and transferred to PVDF membranes (ImmobilonTM, Millipore). Primary rabbit or mouse antibodies were diluted in 5% fat-free dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween?-20. Membranes were incubated with the primary antibody for 1–2 h, followed by incubation for 1 h with either peroxidase-labeled goat anti-mouse IgG or peroxidase-labeled swine anti-rabbit IgG (DAKO, Denmark). Membranes were treated with ECL chemiluminescence reagent (ECLTM, Amersham Biosciences) and the bands visualized by exposing the membranes to HyperfilmTM, ECLTM (Amersham Biosciences).
RESULTS
The primary objective of this study was to isolate UNG2-containing complexes, if present, by immunoprecipitation, and to examine their ability to carry out complete repair of uracil-containing DNA. For this purpose, we used antibodies attached to magnetic beads as bait. The experimental strategy for the repair studies is outlined in Figure 1. As DNA substrate for BER reactions, we used cccDNA containing a single uracil, an AP-site, or a nicked AP-site at a defined position, either opposite of A or G (38,39). The BER type (short-patch or long-patch) was examined after recovery of DNA. Digestion of this DNA substrate with the restriction enzymes XbaI/HincII yields an 8 nt fragment that will only contain label in the one nucleotide at the site of damage (position 0) (short-patch BER). Digestion with HincII/PstI yields an 8 nt fragment that may contain label in positions +3, +4 and +7 (long-patch repair) when using dCTP as radioactive label. Note that while the HincII/PstI cleavage detects long-patch repair exclusively (dCMP incorporated in positions +3 +4 and +7), XbaI/HincII cleavage detects short-patch predominantly, but also the first nucleotide inserted in long-patch repair. The relative contribution of short-patch and long-patch repair is therefore not accurately defined by the assay. Digestion with BamHI/PstI yields a fragment of 22 nt if ends are ligated, and a fragment of 14 nt if the dominant short-patch product is not ligated.
Isolation of functional UNG2-ARC prepared from HeLa extracts
Using PU1sub antibodies attached to paramagnetic beads (PU1sub-beads), we succeeded in isolating complexes proficient in short-patch and long-patch repair of U/G substrate, although short-patch repair was most prominent (Figure 2A). The weaker band of high molecular weight DNA (HMW, theoretically 3191 nt) in Figure 2A represents unspecific incorporation in the rest of the plasmid. The increased strength of the corresponding HMW band in lane 3 is the result of the contribution of short-patch repair in this band, as expected from the HincII/PstI cleavage pattern. As control, we used a substrate with C/G in the same position as U/G. No repair incorporation in the 8 nt fragments was observed with the C/G substrate, and the general HMW background was low, thus the incorporation is damage specific (lanes 2, 4 and 6). We did not succeed in preparing BER-proficient complexes using the other UNG-specific antibodies described in Materials and Methods, or the APE1-specific antibodies (data not shown).
Figure 2. BER carried out by UNG2-ARC. UNG2-ARC was prepared from whole HeLa cell extract and used in BER assays. (A) BER was carried out using U/G substrate or C/G as control and dCTP as radioactive isotope. After incubation, DNA was purified and digested with restriction enzymes to release short- (SP-BER) and long-patch (LP-BER) repair products as well as total repair products (ligated/unligated products, BamHI/PstI). HMW, high molecular weight band. (B) BER as function of time and input of UNG2-ARC. The indicated amount of beads carrying UNG2-ARC was incubated for specified time periods in BER assay. Short-patch BER products were then analyzed and quantified. (B) Results from a single experiment. (C) Results calculated from four independent experiments with error bars showing SD. Gels were analyzed by phosphorimaging.
We then examined BER carried out by UNG2-ARC as function of time and amount of UNG2-ARC present in the incubation. We found an essentially linear dependency of BER with time, and also near-linear dependency on input of beads, except at the highest input of UNG2-ARC. Figure 2B shows results from single experiments, and Figure 2C shows the results calculated from four independent experiments. These results demonstrate that the substrate concentration used is not a limiting factor and that the BER capacity of the complexes is relatively stable under the incubation conditions used. We found that IgG-binding approached saturation when using 400 μg protein extract per 5 μl beads (data not shown), and therefore used these relative amounts of beads and extract routinely for preparation of UNG2-ARC. We also routinely used UNG2-ARC complexes remaining attached to the beads.
We wanted to analyze the proteins captured by PU1sub. For this purpose, we resolved immunoprecipitates by SDS–PAGE and silver-stained the gel (Figure 3A). The lower panel shows BER capacity of the immunoprecipitates. Western analysis demonstrated that PU1sub specifically immunoprecipitated UNG2, POL, PCNA and XRCC1 (Figure 3B). We estimated that <0.05% of the total protein in the extracts were attached to the beads (data not shown). In contrast, total UDG activity was clearly reduced by PU1sub from the same extract (Figure 3C, upper panel). The UDG assay measures activities of UNG1 and UNG2, and the two forms are essentially equally active. By western analysis, we found that approximately 50% of total UNG2, but no appreciable UNG1, were captured with the standard input of PU1sub beads (1x). When bead input was doubled (2x), over 80% of total UNG2 were captured. Therefore, the decreased UDG activity is solely due to extraction of UNG2 by the PU1sub beads (Figure 3C, lower panel). The broad UNG2 band in western analysis (lower panel) is due to different phosphorylated forms of the protein. We also demonstrated that UDG activity is associated with these PU1sub beads (Figure 3D). The weak bands representing repair products seen when using control IgGs (Figure 3A, lower panel) are likely to result from unspecific binding of UNG protein (Figure 3D), and other repair proteins like POL (Figure 3B) to these IgGs. Control experiments demonstrated that essentially equal amounts of UNG2-specific and control IgG were equally efficiently coupled to beads (data not shown). These experiments demonstrate that PU1sub-beads partially deplete extract of UNG2 and associated BER proteins, without measurable reduction in total protein in the extracts. Identification of specific BER factors in the complex by silver staining is complicated by the presence of many proteins in the immunoprecipitate, and the insufficient sensitivity of silver staining relative to the amount extract used in our experiments.
Figure 3. Characterization of UNG2-ARC. (A) UNG2-ARC was immunoprecipitated using PU1sub antibodies and non-immunized IgGs as control. Proteins were resolved on a 12% SDS–polyacrylamide gel and silver-stained (upper panel). The lower panel shows BER capacity of the corresponding immunoprecipitates using U/A- and AP/A substrates. (B) Western analysis of UNG2, PCNA, POL and XRCC1 in immunoprecipitates. (C) Reduction of UDG-activity in cell extracts by immunoprecipitation. ‘Pre-IP’ indicates UDG-activity prior to immunoprecipitation. The lower panel displays the corresponding western analysis of UNG proteins in cell extracts pre-IP and post-IP. (D) UDG activity of PU1sub- and non-immunized IgG beads. 1x, 7.5 μl input of beads; 2x, 15 μl input of beads.
Functional UNG2-ARC isolated using UNG2-specific antibodies contains proteins required for BER of uracil in DNA
Using whole cell extracts of HeLa cells and PU1sub-beads, we isolated UNG2-ARC that repaired U/G substrate, but such a complex could not be prepared using IgGs from pre-immune and non-immunized animals. Neither was repair of AP/G or nicked AP/G substrates observed when using unspecific IgGs for preparing complexes (Figure 4A). This demonstrates the requirement for specific antibodies and supports the view that UNG2-ARC is captured via UNG2. Furthermore, DNase I treatment of the extract did not reduce the yield of functional UNG2-ARC. Thus, the components of UNG2-ARC are not co-precipitated via common DNA molecules (Figure 4A).
Figure 4. Specificity in isolation of functional UNG2-ARC and western analysis of proteins present. (A) UNG2-ARC was prepared/attempted to be prepared using PU1sub coated beads, beads coated with non-immune IgG, or pre-immune IgG, which were all incubated with 400 μg HeLa whole cell extracts. The beads were incubated with BER assay mixture containing dCTP, and U/G, AP/G or AP-nick/G substrate, as indicated. In lane 2, extract was pretreated with 3 U DNase I at 30°C for 30 min prior to UNG2-ARC isolation. Purified DNA was digested for short-patch analysis. (B) Proteins from beads prepared as in (A) were mixed with denaturing loading buffer, heated at 70°C, separated by electrophoresis, blotted and visualized as described in Materials and Methods.
In general, results from western analysis supported the functional assays. We detected UNG2, APE1, PCNA, POL, POL? and XRCC1 in the UNG2-ARC (Figure 4B). We did not detect DNA ligase I possibly because of the properties of the antibody, since we only detected a weak band corresponding to DNA ligase I in whole cell extract using the same antibody. We did not investigate the possible presence of DNA ligase III. We also did not detect APE2, SMUG1, POL, POL, TDG or UNG1 in the UNG2-ARC, although they were all detectable in whole cell extracts (data not shown). The different intensities of the bands do not necessarily reflect the molar ratio of the proteins in the complex, but may merely indicate different properties of the antibodies used for western analysis. As control, we carried out similar experiments using pre-immune IgG linked to beads. No UNG2 was precipitated non-specifically (Figure 3B). The absence of detectable PCNA in the control suggests that PCNA is captured via interaction with immobilized UNG2, with which it is known to interact directly (22). Precipitation of POL was strongly reduced, but not completely absent with pre-immune IgG (Figure 4B). Pre-treatment of the extract with DNase I did not affect the capture of UNG2, PCNA or POL significantly, in agreement with the functional BER assays (Figure 4A).
We also found that proficient UNG2-ARC could not be prepared from an UNG–/– human lymphoblastoid cell line (36), strongly supporting the view that the BER complex is attached via UNG2 (Figure 5A). As an additional control, we successfully prepared proficient UNG2-ARC from peripheral blood lymphocytes, as well as cultured myeloma cells (data not shown). Using whole cell extract from hUNG–/– cells instead of UNG2-ARC, we detected repair of all three DNA lesions, indicating UNG2-independent BER in the extracts (Figure 5A). In agreement with this result, we could not detect any of the BER factors attached to beads coated with PU1sub when we attempted to isolate UNG2-ARC from UNG–/– lymphoblastoid cells (Figure 5B). Furthermore, UNG2 was not detectable in whole cell extracts from these cells, while PCNA, POL, POL? and XRCC1 were all identified (Figure 5B).
Figure 5. BER and western analysis with UNG–/– human lymphoblastoid cells. (A) Whole cell extract was prepared from UNG–/– human lymphoblastoid cells. Whole cell extract (50 μg protein) was used in BER assays with dCTP and substrates as indicated (lanes 4–6), or ‘UNG2-ARC’ was attempted to be prepared from whole cell extracts using PU1sub antibody, and used for BER assays with substrates as indicated (lanes 1–3). (B) Protein in whole cell extract (lane 2) or from immunoprecipitated ‘UNG2-ARC’ (IP) attempted to be prepared from whole cell extract using PU1sub (lane 1) were subjected to western blot analysis as in Figure 4B.
These experiments demonstrate that isolation of functional UNG2-ARC requires interaction between UNG2 and UNG2-specific antibodies and that complex formation is dependent on protein–protein interactions rather than protein–DNA interactions.
BER activity for uracil-containing DNA in UNG2-ARC and whole cell extracts has preference for U/G over U/A
To further examine the BER process in UNG2-ARC, we used a cccDNA substrate containing uracil opposite adenine (U/A) at the same position as in the U/G substrate. This eliminates possible differences related to sequence dependency of the repair process. As demonstrated in Figure 6, we consistently found that repair of U/G was better than repair of U/A, and was not a result of different quality of the substrate as such. Repair of U/A (upper panel) and U/G (lower panel) by UNG2-ARC was abolished by PU101, while a neutralizing anti-SMUG1 antibody (7) had no detectable inhibitory effect. This is consistent with our failure to detect SMUG1 in the complexes. Furthermore, in the presence of PU101, repair of U/A by whole cell extract after 60 min incubation was essentially undetectable (Figure 6, upper panel), and repair of U/G after 10 min was approximately 80% reduced (Figure 6, lower panel). This suggests that UNG2 is the major glycosylase responsible for repair of uracil-containing DNA in U/A pairs and U/G mispairs. However, whole cell extracts carried out a delayed, but significant repair of U/G after prolonged incubation in the presence of PU101 antibodies (Figure 6, lower panel). In whole cell extracts anti-SMUG1 antibodies alone had no significant effect on BER (Figure 5, lane 6), but in the presence of PU101, a small but further increase in inhibition by anti-SMUG1 antibodies was observed, when compared with PU101 alone (Figure 6, lane 8). These results indicate that UNG2 is the main, if not sole, enzyme for repair of U incorporated opposite of A, and the major enzyme for repair of U/G mispairs resulting from deamination of C. Furthermore, SMUG1 may have a back-up function, but is not the primary enzyme responsible for repair of U/G mispairs in this in vitro system. The failure of PU101 antibodies and anti-SMUG1 antibodies to completely inhibit BER when added together may indicate that the two other uracil-DNA glycosylases known, TDG and MBD4, as well as the newly identified DNA glycosylases NEIL1 and NEIL2 (40) may also be considered as potential candidates for the ‘delayed’ U/G repair. Although TDG has very low turnover of substrate alone, the rate is enhanced by APE1 (41), which is certainly present and active both in whole cell extract and in the complexes. Although unlikely because of the small patch size, we could not formally exclude DNA repair mechanisms other than BER in the whole cell extracts; e.g. nucleotide excision repair (NER) or mismatch repair (MMR). However, MMR repair was excluded as a significant source of repair in this system by using nuclear extracts prepared from MMR-deficient HCT-116 colorectal cancer cells (ATCC CCL-247) in the presence of both neutralizing antibodies, which again did not abolish repair completely (data not shown). Finally, these results indicate that UNG2 is the only uracil-DNA glycosylase present in UNG2-ARC.
Figure 6. Roles of UNG2 and SMUG1 in short-patch repair of U/A pairs and U/G mispairs. UNG2-ARC or whole cell extract of HeLa cells was incubated with BER assay mixture for 60 min with dTTP and U/A (upper panel), or with dCTP and U/G substrate (lower panel) in the absence or presence of anti-UNG antibody (PU101) or anti-SMUG1antibody (PSM1) or both, as indicated. The diagram represents results from four independent experiments where bars indicate SD.
Inhibition of UNG2 does not impair subsequent BER steps and recombinant SMUG1 partially restores BER of U/G by UNG2-ARC, but not U/A repair
To test whether inhibition of UNG2 affects subsequent BER steps carried out by UNG2-ARC, we assayed repair of U/G, AP/G and nicked-AP/G DNA substrates in the presence of neutralizing PU101 antibody. The antibodies completely inhibited repair of U/G, whereas AP/G and nicked AP/G substrates were effectively repaired (Figure 7A). Addition of 2 ng of recombinant SMUG1 enzyme (7) to PU101-inhibited UNG2-ARC, partially restored BER capacity for U/G and this restoration was inhibited by neutralizing SMUG1 antibodies, as expected. Next, we tested repair of U/A substrate under similar assay conditions. Neutralizing PU101-antibodies also inhibited repair of U/A completely, while repair of AP/A and nicked AP/A was not affected (Figure 7B). Contrary to experiments with U/G substrate, 2 ng recombinant SMUG1 enzyme did not restore BER of U/A. SMUG1 was previously shown to remove U from U/A pairs, although kcat/Km is 3.2-fold higher for U/G than for U/A in uracil release assays (7). These findings may in part explain the inability of SMUG1 to complement UNG2 in repair of U/A pairs.
Figure 7. Lack of functional coupling between the glycosylase step and the subsequent steps in short-patch repair. (A) UNG2-ARC was incubated with BER assay mixture with dCTP and U/G, AP/G or AP-nick/G as indicated, in the absence or presence of anti-UNG antibody (PU101) and/or anti-SMUG1 antibody (PSM1), and absence or presence of 2 ng recombinant SMUG1 (rec. SMUG1). (B) UNG2-ARC was incubated as in (A), but with dTTP and U/A, AP/A or AP-nick/A substrate.
APE1 is indispensable for BER activity and immunodepletion of XRCC1 reduces BER capacity of UNG2-ARC and the yield of ligated repair product
Mammalian cells have two AP-endonucleases, APE1 and APE2, that share homology with Escherichia coli exonuclease III (ExoIII). To test the relative contribution of these to the AP-endonuclease activity of UNG2-ARC, we conducted BER assays in the absence or presence of polyclonal anti-APE1 antibodies. We found that APE1 antibodies completely inhibited BER activity of UNG2-ARC (Figure 8A), indicating that APE1 is the only functional AP-endonuclease in UNG2-ARC. Further, we tested the functional role of XRCC1 in UNG2-ARC, and found that polyclonal antibodies to XRCC1 had no direct inhibitory effect in BER (Figure 8A). However, immunodepletion of XRCC1 resulted in markedly reduced BER, and a significant fraction of repaired products remained unligated after XRCC1 depletion (Figure 8B). These results are consistent with the presumed role of XRCC1 as scaffolding protein that interacts with other BER proteins, including POL?, DNA ligase III, and the fact that XRCC1 itself has no known catalytic activity.
Figure 8. Roles of APE1 and XRCC1 in BER. (A) UNG2-ARC was incubated with BER assay mixture in the absence or presence of APE1 and XRCC1 polyclonal antibodies as indicated. (B) Whole HeLa extracts were incubated with XRCC1-antibody linked beads (lanes 2 and 4) or non-immunized serum IgG-linked beads (lanes 1 and 3) for 4 h at 4°C. The beads were removed and the extracts were further incubated with PU1sub-linked beads. The UNG2-ARC thus captured was subsequently used in BER assay.
UNG2-ARC isolated from growth-arrested HaCaT cells display lower BER activity than UNG2-ARC isolated from exponentially growing cells
To examine BER activity of UNG2-ARC in relation to proliferative status of the cells, we isolated complexes from cell extracts prepared from exponentially growing cells (EC) and growth-arrested (AC) HaCaT cells. The cell cycle status of the cells was determined by FACS analysis (Figure 9A). Using the BER assay, we found a markedly lower BER activity (short- and long-patch) after 15 min in UNG2-ARC isolated from growth-arrested HaCaT cells compared with those prepared from exponentially growing cells (Figure 9B and C, lanes1 and 2, and diagram E). However, after 60 min incubation the BER process had reached equal levels of short-patch repair in the complexes (Figure 9B, lane 4 and 5, and diagram E), whereas long-patch repair of UNG2-ARC isolated from arrested cells was only 30% of that from growing cells (Figure 9C, lane5 and 6, and diagram E). This is consistent with the roles of several DNA replication factors in long-patch BER, implying close association of these DNA metabolizing systems. Furthermore, using the BER assay, we found similar patterns of repair in the corresponding whole cell extracts (data not shown). Addition of neutralizing anti-POL? antibodies to the BER mixture resulted in stronger inhibition of repair activity of UNG2-ARC prepared from the growth-arrested cells compared with that from growing cells (Figure 9B and C, compare lanes 4 and 5 with lanes 7 and 8, respectively, and diagram E). This indicates that POL? is the predominant repair DNA polymerase in the UNG2-ARC isolated from arrested cells. Adding 3-fold higher amounts of neutralizing anti-POL? antibodies to the repair reaction had no additional inhibitory effects (data not shown). In a previous study, inhibition of POL? was found to reduce long-patch by nearly 75% in cell extract, indicating involvement of POL? in this pathway (42). This agrees well with our results demonstrating reduction of long-patch BER when POL? activity was inhibited by neutralizing antibodies (Figure 9C, lanes 7 and 8). This indicates that either a fraction of long-patch repair in UNG2-ARC is initiated by POL? (10,43) or that POL? may synthesize repair patches longer than one nucleotide (11).
Figure 9. BER activity in proliferating and non-proliferating cells. UNG2-ARC was isolated from whole extract of exponentially growing HaCaT cells (EC) as well as from whole extract of high-density and growth-arrested HaCaT cells (AC) that did not replicate DNA. (A) Comparison of DNA content distributions as analyzed by flow cytofluorometry analysis of propidium iodide stained HaCaT cells. Grey histogram; exponentially growing proliferating cells, white histogram; growth-arrested non-proliferating cells. Fluorescence intensity is on the linear axis, while the vertical axis indicates the relative number of stained cells. UNG2-ARC was isolated from the extracts and used in BER assays with U/A substrate and dTTP isotope and incubated for indicated time periods in the absence (lanes 1–6), or presence (lanes 7–8) of neutralizing POL? antibodies. The repair products were purified and digested with restriction enzymes to release short-patch (B) and long-patch (C) repair products as well as total repair products (ligated/unligated products, BamHI/PstI) (D). Results calculated from two independent experiments, each in duplicate, for BER kinetics analysis of exponentially growing cells relative to growth-arrested HaCaT cells after 15 min incubation and in the presence and absence of anti-POL? antibodies after 60 min BER incubation (E).
The capacity of BER activity of cell extracts and UNG2-ARC is rate-limited not solely by the cellular content of UNG2
Base excision repair proteins has been suggested to be sequentially recruited to the site of repair, implying that repair complexes are generated in proportion with damage to DNA (44). Alternatively, the buildup of repair complexes such as UNG2-ARC could depend on the concentration of repair factors. For this purpose, we prepared extracts from ordinary HeLa cells and stably transfected HeLa cells overexpressing UNG2 (HTO-UNG2-45). We found 15-fold higher level of UDG enzymatic activity in induced cells compared with non-induced cells (data not shown). To examine uracil BER capacity in extracts and UNG2-ARC, we used U/A substrate because repair of this lesion is completely dependent on functional UNG, in contrast with U/G repair (Figure 6). The rate of uracil repair was increased 4-fold in cell extract prepared from induced cells compared with non-induced cells at 5 and 10 min incubation (Figure 10, lanes 1–6 and the panel below). The increase in BER capacity of UNG2-ARC isolated from UNG2-overexpressing cells was smaller (1.5- to 2.5-fold, lanes 7–10 and the panel below). The data from crude extracts indicate that UNG2 may be a rate-limiting factor, but since the BER rate increases 4-fold when UNG2 increases15-fold, it does not seem to be the sole rate-limiting factor. The results of BER analysis of UNG2-ARC indicate that formation of repair complexes could be independent of the presence of damage in DNA.
Figure 10. Increased cellular UNG2 protein enhances BER activity in whole cell extract and UNG2-ARC. Exponentially growing HeLa cells carrying a UNG2 expressing construct were either induced with 2 μg/ml of doxycycline for 48 h (+) or mock-induced (–) as control. UNG2-ARC was prepared by incubating 40 μl of PU1sub-linked beads with 100 μg of whole cell extract for each reaction. BER assays with 500 ng of U/A DNA substrate and dTTP as isotope were carried out using whole cell extracts (40 μg) (lanes 1–6) or UNG2-ARC (lanes 7–18) for the indicated incubation times. The panels below show the results calculated from two independent experiments.
DISCUSSION
Our work demonstrates the existence of a BER complex that is proficient in repair of uracil-containing DNA and western analysis confirmed that the factors known to be required for the process were present in the complex. Furthermore, the buildup of the complex is not dependent on the presence of uracil-damaged DNA. This indicates that at least one form of BER for uracil-containing DNA takes place in a preassembled multi-protein complex. Alternative models assuming sequential recruitment of enzymes to the site of repair have been suggested (45). Several observations, particularly the pattern of protein–protein interactions, support this model in which an incoming protein is recruited by one or more proteins present at the site of repair. After release of the base in the initial step, the DNA glycosylase may remain bound to the product DNA (AP-site) for which it has high affinity, and may shield it from damage (45). In some cases, DNA glycosylases have virtually no turnover (TDG), or low turnover (SMUG1), when present alone, but the turnover is enhanced several-fold by AP-endonuclease (7,41). The structure of APE1 bound to abasic DNA showed that APE1 uses a rigid, pre-formed surface to bend the DNA helix by 35° and retains its kinked DNA product efficiently (46). The kinked APE1–DNA complex is proposed to recruit POL? to sites of damage (46,47), where it binds to the nicked DNA, and induces a further bend to 90° (47). DNA kinking could also provide an effective means of promoting the directional and sequential exchange of factors at each step of the BER pathway (46). This recruitment model does not require the formation of a more stable BER complex, but is not incompatible with the preassembled BER complex model, where the substrate may be handed from one component of the complex to the other. One possibility is that these mechanisms operate in different contexts. Some components may be recruited as single proteins and others as small complexes, forming larger complexes with limited lifetime. Another possibility, in agreement with our results, is that the biochemistry of the individual steps operates just as suggested for the sequential recruitment model, but that this process simply takes place in a complex.
In a previous paper, a multi-protein BER complex from bovine testis containing POL?, DNA ligase I and uracil-DNA glycosylase was isolated by a POL? affinity method, and if proteins were present in equimolar concentrations, the complex had a cumulative mass of 196 kDa (20). This complex was captured in the absence of DNA by POL?-affinity chromatography. This argues against damage recognition by the glycosylase being a trigger for complex assembly. The fact that BER complexes can be isolated by two different methods supports the existence of such complexes in the cell. However, our work indicates that PCNA, POL, XRCC1, a DNA ligase and APE1 are also part of a BER complex, which must then be significantly larger than the complex reported previously. Possibly, these differences may be explained by the fact that we used lower concentrations of monovalent salt during isolation and washing of the complex. Since we did not detach the BER complex(es) from the beads, and did not examine them as complexes in solution, it is possible that UNG2 is present in complexes with different compositions. In NER, at least some repair factors are recruited sequentially to the site of damage after localized subnuclear exposure to ultraviolet light. Here the damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors (48). Thus, at best a fraction of NER proteins is present in preassembled repair complexes.
Based on experiments using isolated nuclei (22) and Ung-deficient mice (49), a role for UNG2 in removal of misincorporated uracil in U/A pairs has been established. However, the roles of UNG2, SMUG1 or other enzymes, in removal of uracil from U/G mispairs have remained unsettled (7,49). We find that UNG2-ARC and whole cell extracts are at least as effective in repair of U/G mispairs, as with U/A pairs. Previously, we defined kinetic properties of homogenous recombinant SMUG1 and UNG2 using U/A or U/G substrates in oligonucleotide cleavage assays. We concluded that these properties were consistent with a major role for UNG2 in repair of U/G mispairs, as well as U/A pairs. Complete BER in crude extracts of HeLa cells supported this view, in agreement with results presented here (7). However, a different paper concluded that murine SMUG1 has the major uracil-DNA glycosylase activity initiating repair on U/G mispairs, with UNG2 as a back-up (50). In these studies, the glycosylase step was examined using nuclear extracts in oligonucleotide cleavage studies, and low substrate concentrations were used to mimic the in vivo situation. The different conclusions reached in the latter study and the present study are not due to differences in the concentration of DNA-uracil, which are fairly equal in the two studies. It could, however, possibly be due to a much higher total DNA concentration but much lower relative lesion density used in our study as compared with study on murine extracts (50). The different results are, however, more probably explained by the use of different methods for preparation of extract, and by different kinetics due to assay conditions. It should also be noted that activities and kinetic properties of human UNG2 (7) and human and mouse SMUG1 (7,49) are dependent on concentrations of monovalent salts and Mg2+, as well as the presence or absence of APE1. Contrary to the kinetic properties originally reported for Xenopus SMUG1, and the erroneous entries of kinetic parameters for hUNG in this paper (51), kinetic parameters for human SMUG1 and UNG2 would be consistent with a major role for UNG2 and a backup function for SMUG1 in removal of uracil from DNA both in U/G mispairs and U/A pairs (7,52). Thus, the Km values of SMUG1 and UNG2 are 1.3 μM and 0.4 μM, respectively, whereas Kcat for UNG2 is 100-fold higher than that of SMUG1 and Kcat/Km some 300-fold higher than that of SMUG1 (7). Even if different assay conditions may modify the picture several-fold, the properties of SMUG1 does not make it a likely primary candidate in performing the important first step in BER of uracil-containing DNA. Our present data, both with BER complexes and whole cell extracts, supports this view. Furthermore, murine (53) and human UNG2 (36) have been demonstrated to be required for removal of uracil in U/G pairs resulting from deamination by AID in somatic hypermutation and class switch recombination in B-cells. The major role of SMUG1 is more likely to be in repair of oxidative base lesions, such as 5-hydroxymethyluracil (7,52,54) 5-hydroxyuracil and 5-formyluracil (52), where alternative repair enzymes are not identified, or are less efficient. Thus, we conclude that UNG2 is the major enzyme initiating BER of uracil-containing DNA both in a U/G and a U/A context. UNG2-ARC also proficiently repair abasic sites, but whether this type of complex represents the major type, completing repair initiated by glycosylases other than UNG2 remains to be examined.
Components of short- and long-patch BER have been identified and their roles studied both in vitro and in vivo as well as in reconstitution systems. However, mechanisms that determine the branching of BER pathway into short-patch and long-patch repair are not completely understood. The type of DNA damage was found to determine the mode of pathway (55). Thus, repair of hypoxantine (HX) and 1,N6-ethenoadenine (A) by the monofunctional alkyl-N-purine glycosylase (ANPG/MPG) was via both short- and long-patch BER, whereas repair of 8-oxo-G by the bifunctional hOOG1 protein was mainly via short-patch pathway (55). However, in a recent study on repair of 8-oxo-G, 55–80% repair patches were >1 nt in an in vivo system (56). However, the biochemistry of the repair pathway was not analyzed in this study, nor was possible impact of cell cycle status on the mode of repair. Modified 5'-dRp residues are refractory to the dRPase activity of POL? (57). Thus, repair DNA synthesis of such lesions could be initiated by POL? (58), but further removal of the 5'-dRp and completion of repair is thought to require a ‘switchover’ to a PCNA and POL/ dependent long-patch pathway (59). Although single nucleotide insertion is a dominant function of POL?, this DNA polymerase is able to replace several nucleotides containing a modified 5'-dRp, resulting in the generation of a flap oligonucleotide structure (11,60). The flap is cleaved by FEN1 (11), resulting in a nick in DNA which is subsequently sealed by a DNA ligase. A direct involvement of PCNA in BER outside sites of replication has not so far been demonstrated. However, a PCNA-independent long-patch BER outside sites of replication consisting of a core of four proteins; APE1, POL?, FEN1 and DNA ligase III may occur. Such long-patch repair might be the main mode of BER of modified AP-sites in resting cells. This model may be consistent with previous reports (11,42) as well as our data. The rate-limiting step in BER has previously been studied and suggested to be the removal of 2-deoxyribose 5-phosphate (dRp) when BER is initiated by a monofunctional glycosylase (e.g. UNG) (61), or the removal of 3'-blocking deoxyribose residue when a base damage is removed by a bifunctional DNA glycosylase (e.g. hOGG1) (62). Moreover, in a separate study, the joining of repair ends by a DNA ligase was suggested to be a rate-limiting step in BER (63,64). Although the rate of excision of uracil from DNA by UNG-proteins is highly sequence dependent (35), UNG2 and other UNG-proteins have very high catalytic turnover numbers (7,65). This suggests that glycosylase step is not the most likely rate-limiting step in UNG2-ARC and probably not in extracts. Previously, the excision of certain base lesions was also found to be a rate-limiting step in BER when a DNA glycosylase with low turnover activity removed the lesions (66). Apparently, the type and the position of a damaged base are additional factors in the kinetics of BER. In fact, we found that placing uracil opposite adenine at the neighboring nucleotide at the 5'-side of the U/A substrate used in our experiments (Figure 1) resulted in a significant reduction of rate of repair (data not shown).
In conclusion, we immunoprecipitated BER complexes that carry out short-patch and long-patch BER for uracil-containing DNA, using antibodies specific for the N-terminal non-catalytic part of UNG2. Short-patch repair is the predominant mode both in extracts and in BER complexes. Furthermore, UNG2 appears to be the major glycosylase responsible for repair of U/G mispairs and possibly sole enzyme for repair of U/A pairs in human cells. On the basis of the results presented here, we suggest that at least a significant fraction of BER proteins for repair of uracil-containing DNA, and possibly for other BER substrates as well, are present in preassembled repairosome multiprotein BER complexes. These may be dynamic of nature.
ACKNOWLEDGEMENTS
This work was supported by The Research Council of Norway, The Norwegian Cancer Society, The Cancer Fund at St Olav's Hospital, Trondheim, and The Svanhild and Arne Must Fund for Medical Research, INSERM and the European Economic Community (contract number QLG1-CT-2001-01536- IMPAD). J.P.-D. is a recipient of a Marie Curie fellowship from the European Community programme IHP-MCIF-99-1 under contract number HPMF-CT-2001-01290.
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* To whom correspondence should be addressed. Tel: +47 73598695; Fax: +47 73598801; Email: hans.krokan@medisin.ntnu.no
ABSTRACT
Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POL?, POL, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POL?, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POL? dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).
INTRODUCTION
Uracil in DNA occurs as a result of deamination of cytosine and incorporation of dUMP during replication. Deamination of cytosine occurs at a rate of 100–500 per human cell per day, yielding mutagenic U/G mispairs which, unless repaired, result in GC to AT transitions upon replication (1). Incorporation of dUMP during replication results in U/A pairs which are not miscoding, but which may yield cytotoxic and potentially mutagenic abasic (AP) sites (2). Uracil in DNA may also affect transcriptional fidelity (3), as well as binding of transcription factors (4). A recently identified source of uracil in the genome is the enzymatic deamination of cytosine to uracil by activation-induced cytidine deaminase (AID) in the process of somatic hypermutation and antibody class switch in B-cells (5). Uracil is recognized by a uracil-DNA glycosylase (UDG) activity, which cleaves the N-glycosylic bond leaving an AP-site in DNA. Human cells contain at least four types of UDG; mitochondrial UNG1 and nuclear UNG2, SMUG1, TDG and MBD4, which have overlapping substrate specificities (6). Their specific functions are still unclear. Among these glycosylases, UNG proteins are by far the catalytically most efficient (6,7). UNG1 and nuclear UNG2 are both encoded by the UNG-gene and have a common catalytic domain, but different N-terminal sequences required for subcellular sorting (8). Upon mitochondrial import the preform of UNG1 is processed to a mature form lacking 29 N-terminal amino acid residues (9). AP-sites in nuclear DNA are repaired by either single-nucleotide (short-patch) base excision repair (BER) or via replacement of several nucleotides (long-patch BER). Short-patch BER requires an AP endonuclease, POL? and DNA ligase III or possibly also DNA ligase I, while long-patch BER depends on flap endonuclease I (FEN-1), and may require proliferating cell nuclear antigen (PCNA), DNA polymerases / and DNA ligase I. POL? has also been suggested to be required for long-patch repair either for insertion of the first nucleotide (10) or for strand displacement (11). An APE1-independent short-patch BER pathway has recently been suggested. Thus, the bifunctional DNA glycosylases NEIL1 and NEIL2 carry out ?, -elimination after excision of damaged bases generating a one-nucleotide gap flanked by 3'-phosphate and 5'-phosphate termini. The 3'-phosphate terminus may subsequently be converted to a 3'-OH terminus by polynucleotide kinase (PNK), thus setting up for short-patch repair (12).
In DNA repair and related processes, there is evidence for the existence of functional multi-protein complexes. Thus, multi-protein complexes involved in eukaryote transcription and replication have been reported (13,14). A group of proteins that associates with DNA repair protein BRCA1 has been identified and named BRCA1-associated genome surveillance complex (BASC). It includes DNA repair proteins MSH2, MSH6, MLH1, ATM, BLM and the RAD50–MRE11–NBS1 protein complex, PCNA and RF-C (15). Furthermore, a multi-protein complex that connects Fanconi anemia and Bloom syndrome has recently been reported (16). Many interactions between the proteins in the initial step of nucleotide excision repair (NER) have also been reported and the existence of a multiprotein repairosome complex was suggested (17), although a later study did not find evidence in support of such a complex (18). Nevertheless, it is evident that many interactions do occur and it may seem likely that the mechanism of NER at least in part relies on formation of complexes (19). There is also evidence for complex formation in BER. Thus, a 180 kDa protein complex that repairs uracil-containing DNA was isolated from bovine testis using DNA polymerase ?-affinity chromatography (20). Direct molecular interactions between UNG2 and other BER factors, e.g. RPA (21) and PCNA (22) have also been reported. However, PCNA interacts with multiple partners, e.g. POL, POL, RF-C, DNA ligase I, FEN-1, POL?, APE1 and XRCC1, all of which are involved in long-patch BER, and all, except POL? and XRCC1, in DNA replication (23–25). Most likely PCNA engages in different complexes, but it is also possible that the three binding sites in this homotrimeric protein bind different proteins. POL? also interacts directly with ligase I (26), XRCC1 (27) and APE1 (28). In addition, XRCC1 interacts with DNA ligase III (27,29) with poly(ADP-ribose) polymerase (PARP) through one of its BRCT-modules (30), PNK (31), APE1 (32) and PCNA (24) acting as a scaffold protein in BER and single-strand break repair (33).
Given the plethora of interactions between BER proteins, it seemed likely that it would be possible to isolate a complex for nuclear BER of uracil-containing DNA, using a UNG2-specific antibody or other BER-antibodies. The present work demonstrates the presence and properties of an UNG2-containing protein complex proficient in complete repair of uracil-containing DNA.
MATERIALS AND METHODS
Synthetic oligonucleotides were from MedProbe (Oslo, Norway). dTTP and dCTP (3000 Ci/mmol) were from Amersham Biosciences. Primary antibodies against POL (mouse monoclonal) were from Transduction Laboratories, Lexington, KY, antibodies against PCNA (ab29, PC10), POL? (mouse monoclonal) and XRCC1 (rabbit polyclonal) were from Abcam Ltd, UK, and antibodies against APE1 (rabbit polyclonal) from Novus Biologicals Inc., Littleton, CO, USA. Rabbit antiserum to APE2 was kindly provided by Magnar Bj?r?s (Centre of Molecular Biology and Department of Molecular Biology, Oslo). Human recombinant POL? and neutralizing antibodies to POL? were generous gifts from Dr S. H. Wilson (Laboratory of Structural Biology, NIH). Paramagnetic Protein A-beads were from Dynal, Oslo, Norway. A neutralizing polyclonal rabbit anti-hSMUG1 IgG (PSM1) was prepared as described previously (7). Silver-staining of protein gels was carried out using the ProteosilverTM Plus, Silver Stain Kit (Sigma-Aldrich Co.).
DNA substrates
Covalently closed circular DNA (cccDNA) substrates were prepared essentially as described previously (34). Briefly, 20 μg of ssDNA were annealed to 4.2 μg of a 5' phosphorylated 22mer complementary oligonucleotide containing either a uracil or normal base (molar ratio 1:30) (Figure 1A). Synthesis of duplex DNA was carried out in the presence of T4 DNA polymerase, T4 DNA ligase and T4 gene 32 ssDNA-binding protein at 37°C for 2 h. cccDNA duplex molecules were purified by CsCl gradient centrifugation. The purity of the substrate prepared was estimated by agarose gel electrophoresis (Figure 1B). Only DNA substrate containing >95% cccDNA was used in BER assays. By site-directed mutagenesis of a pGEM-3zf(+) Phagemide, we prepared a G to A mutation to be able to prepare substrates containing U/A and U/G in the same sequence context. For AP-site substrate, uracil-containing cccDNA was incubated with purified catalytic domain of UNG (35) just before use. Complete removal of uracil was confirmed by further incubation of an aliquot of this DNA with purified APE1 protein resulting in complete conversion of form I (supercoiled closed circular DNA) to form II (nicked circular DNA) detected by agarose gel electrophoresis (data not shown). For generation of AP-nick substrate DNA, uracil-containing plasmid was incubated with purified catalytic domain of UNG and purified APE1 protein.
Figure 1. Substrate and strategy for BER studies. (A) As substrate for the BER assay we used double-stranded covalently closed circular pGEM-3Zf(+) with U/G, U/A, AP-site/G or nicked AP-site/A in the same position. The cleavage sites for restriction enzymes used for mapping of the repair patches are indicated. The shaded region corresponds to the repaired patch. (B) The quality of the substrate in terms of the amount of covalently closed circular plasmid DNA (form I) and nicked circular forms (form II) purified by CsCl/ethidium bromide equilibrium centrifugation. Purity was assessed by running 100 ng of DNA on 1% agarose gel with PstI digested DNA as marker. (C) Specificity of anti-UNG-antibodies PU101 and PU1sub. Recombinant UNG2, the mitochondrial form UNG129 and the catalytic UNG-domain were separated by polyacrylamide gel electrophoresis and subjected to western analysis using either PU101 or PU1sub, as indicated. (D) Structure of the preform of UNG1, UNG129, UNG2 and the catalytic domain UNG84.
UNG antibodies
Neutralizing anti-UNG-antibody PU101 against the catalytic domain has been described previously (35). Polyclonal anti-UNG2 PU1 was prepared following the same procedure. PU1sub antibody against the N-terminal region of the human UNG2 was prepared by passing PU1 IgG over a matrix containing the recombinant C-terminal catalytic UNG-domain bound to NHS-activated Sepharose (Amersham Biosciences). The IgGs in the flow-through proved to be specific for the UNG2 N-terminal regulatory domain as judged from western analysis (Figure 1C and D), and did not inhibit UNG enzyme activity.
HeLa Tet-On cells (HTO) overexpressing UNG2 (HTO-UNG2)
UNG2 cDNA was cloned into the EcoRI/XbaI sites of vector pTRE and the construct (pTRE-UNG2) co-transfected with pTK-Hyg into HTO cells. Hygromycin resistant clones were selected and subcloned by dilution. The subclone that repeatedly gave the best expression after induction, HTO-UNG2-45, was used in the present study.
Culture of cell lines and preparation of whole cell extracts
HaCaT, HeLa S3 and HTO-UNG2 cells were cultured in DMEM with 10% fetal calf serum (FCS), 0.03% glutamine and 0.1 mg/ml gentamicin at 5% CO2. Human myeloma cell line JJN-3 was cultured under similar conditions but in RPMI 1640 medium. Peripheral blood lymphocytes were obtained by density gradient centrifugation of buffy coat over LymphoprepTM (Nycomed, Norway). The UNG–/– lymphoblastoid cell line was from patient 2 (36) and carried a Phe251Ser homozygous mutation. Cells were grown in RPMI 1640, with 0.03% glutamine, 10% heat-inactivated FCS, and 100 U/ml penicillin and 100 μg/ml streptomycin at 5% CO2.
Whole cell extracts were prepared essentially as described by Tanaka et al. (37). Briefly, cells were pelleted at 215 g and resuspended at 1x packed cell volume in buffer I and 1x packed cell volume of buffer II . The mixture was rocked at 4°C for 2 h and cell debris was pelleted at 22 000 g at 4°C for 10 min. The supernatant was recovered and protein concentration measured using the Bio-Rad protein assay. Extracts were snap frozen in liquid nitrogen and stored in small aliquots at –80°C.
Preparation of BER complex UNG2-ARC
PU1sub IgGs were covalently linked to magnetic Dynabeads? Protein A using dimethyl pimelimidate dihydrochloride (DMP) according to instructions from the manufacturer (Dynal, Norway) with minor modifications: 400 μg protein from whole HeLa cell extract was mixed with 5 μl of the antibody-coated beads or otherwise indicated, and kept in suspension under constant and gentle rocking for 4 h at 4°C. The beads were washed three times with 10 mM Tris–HCl, pH 7.5, transferred to a new tube, washed once more in the same buffer and resuspended in appropriate buffer for further use. For control experiments, we prepared beads linked to the same amount of pre-immune IgG from the same rabbit (pre-immune-IgG), and non-immunized rabbit serum (non-immune-IgG).
BER assay
The BER mixtures (50 μl) contained (final) 40 mM HEPES–KOH (pH 7.8), 70 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 2 mM ATP, 20 μM dATP, 20 μM dGTP, 8 μM dCTP or dTTP depending on the isotope used, 4.4 mM phosphocreatine, 2.5 μg creatine kinase, 18 μg BSA, 2μCi dCTP or dTTP, 50 μg whole cell extract (measured as protein) or UNG2-ARC (5 μl beads) or otherwise immunoprecipitated material, and 300 ng cccDNA substrate if not otherwise indicated. For BER assay experiments, the beads were resuspended in 10 mM Tris–HCl, pH 8.0 containing Complete? protease inhibitor and 7% glycerol (final). The repair mixtures were incubated at 30°C for the indicated times (usually 60 min), and stopped by adding (final) 20 mM EDTA and 80 ng/μl RNase A (37°C, 10 min), and further incubated with (final) 190 ng/μl proteinase K and 0.5% SDS (37°C, 30 min). The repair products were purified by phenol/chloroform extraction and ethanol/salt precipitation. DNA was digested with indicated restriction enzymes (Figure 1), and analyzed by electrophoresis in 12% denaturing polyacrylamide gels and phosphorimaging (Fuji, BAS-1800II) of the dried gels.
Western analysis
Proteins were separated on pre-cast 10% denaturing NuPAGE gels (InvitrogenTM, Life Technologies) and transferred to PVDF membranes (ImmobilonTM, Millipore). Primary rabbit or mouse antibodies were diluted in 5% fat-free dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween?-20. Membranes were incubated with the primary antibody for 1–2 h, followed by incubation for 1 h with either peroxidase-labeled goat anti-mouse IgG or peroxidase-labeled swine anti-rabbit IgG (DAKO, Denmark). Membranes were treated with ECL chemiluminescence reagent (ECLTM, Amersham Biosciences) and the bands visualized by exposing the membranes to HyperfilmTM, ECLTM (Amersham Biosciences).
RESULTS
The primary objective of this study was to isolate UNG2-containing complexes, if present, by immunoprecipitation, and to examine their ability to carry out complete repair of uracil-containing DNA. For this purpose, we used antibodies attached to magnetic beads as bait. The experimental strategy for the repair studies is outlined in Figure 1. As DNA substrate for BER reactions, we used cccDNA containing a single uracil, an AP-site, or a nicked AP-site at a defined position, either opposite of A or G (38,39). The BER type (short-patch or long-patch) was examined after recovery of DNA. Digestion of this DNA substrate with the restriction enzymes XbaI/HincII yields an 8 nt fragment that will only contain label in the one nucleotide at the site of damage (position 0) (short-patch BER). Digestion with HincII/PstI yields an 8 nt fragment that may contain label in positions +3, +4 and +7 (long-patch repair) when using dCTP as radioactive label. Note that while the HincII/PstI cleavage detects long-patch repair exclusively (dCMP incorporated in positions +3 +4 and +7), XbaI/HincII cleavage detects short-patch predominantly, but also the first nucleotide inserted in long-patch repair. The relative contribution of short-patch and long-patch repair is therefore not accurately defined by the assay. Digestion with BamHI/PstI yields a fragment of 22 nt if ends are ligated, and a fragment of 14 nt if the dominant short-patch product is not ligated.
Isolation of functional UNG2-ARC prepared from HeLa extracts
Using PU1sub antibodies attached to paramagnetic beads (PU1sub-beads), we succeeded in isolating complexes proficient in short-patch and long-patch repair of U/G substrate, although short-patch repair was most prominent (Figure 2A). The weaker band of high molecular weight DNA (HMW, theoretically 3191 nt) in Figure 2A represents unspecific incorporation in the rest of the plasmid. The increased strength of the corresponding HMW band in lane 3 is the result of the contribution of short-patch repair in this band, as expected from the HincII/PstI cleavage pattern. As control, we used a substrate with C/G in the same position as U/G. No repair incorporation in the 8 nt fragments was observed with the C/G substrate, and the general HMW background was low, thus the incorporation is damage specific (lanes 2, 4 and 6). We did not succeed in preparing BER-proficient complexes using the other UNG-specific antibodies described in Materials and Methods, or the APE1-specific antibodies (data not shown).
Figure 2. BER carried out by UNG2-ARC. UNG2-ARC was prepared from whole HeLa cell extract and used in BER assays. (A) BER was carried out using U/G substrate or C/G as control and dCTP as radioactive isotope. After incubation, DNA was purified and digested with restriction enzymes to release short- (SP-BER) and long-patch (LP-BER) repair products as well as total repair products (ligated/unligated products, BamHI/PstI). HMW, high molecular weight band. (B) BER as function of time and input of UNG2-ARC. The indicated amount of beads carrying UNG2-ARC was incubated for specified time periods in BER assay. Short-patch BER products were then analyzed and quantified. (B) Results from a single experiment. (C) Results calculated from four independent experiments with error bars showing SD. Gels were analyzed by phosphorimaging.
We then examined BER carried out by UNG2-ARC as function of time and amount of UNG2-ARC present in the incubation. We found an essentially linear dependency of BER with time, and also near-linear dependency on input of beads, except at the highest input of UNG2-ARC. Figure 2B shows results from single experiments, and Figure 2C shows the results calculated from four independent experiments. These results demonstrate that the substrate concentration used is not a limiting factor and that the BER capacity of the complexes is relatively stable under the incubation conditions used. We found that IgG-binding approached saturation when using 400 μg protein extract per 5 μl beads (data not shown), and therefore used these relative amounts of beads and extract routinely for preparation of UNG2-ARC. We also routinely used UNG2-ARC complexes remaining attached to the beads.
We wanted to analyze the proteins captured by PU1sub. For this purpose, we resolved immunoprecipitates by SDS–PAGE and silver-stained the gel (Figure 3A). The lower panel shows BER capacity of the immunoprecipitates. Western analysis demonstrated that PU1sub specifically immunoprecipitated UNG2, POL, PCNA and XRCC1 (Figure 3B). We estimated that <0.05% of the total protein in the extracts were attached to the beads (data not shown). In contrast, total UDG activity was clearly reduced by PU1sub from the same extract (Figure 3C, upper panel). The UDG assay measures activities of UNG1 and UNG2, and the two forms are essentially equally active. By western analysis, we found that approximately 50% of total UNG2, but no appreciable UNG1, were captured with the standard input of PU1sub beads (1x). When bead input was doubled (2x), over 80% of total UNG2 were captured. Therefore, the decreased UDG activity is solely due to extraction of UNG2 by the PU1sub beads (Figure 3C, lower panel). The broad UNG2 band in western analysis (lower panel) is due to different phosphorylated forms of the protein. We also demonstrated that UDG activity is associated with these PU1sub beads (Figure 3D). The weak bands representing repair products seen when using control IgGs (Figure 3A, lower panel) are likely to result from unspecific binding of UNG protein (Figure 3D), and other repair proteins like POL (Figure 3B) to these IgGs. Control experiments demonstrated that essentially equal amounts of UNG2-specific and control IgG were equally efficiently coupled to beads (data not shown). These experiments demonstrate that PU1sub-beads partially deplete extract of UNG2 and associated BER proteins, without measurable reduction in total protein in the extracts. Identification of specific BER factors in the complex by silver staining is complicated by the presence of many proteins in the immunoprecipitate, and the insufficient sensitivity of silver staining relative to the amount extract used in our experiments.
Figure 3. Characterization of UNG2-ARC. (A) UNG2-ARC was immunoprecipitated using PU1sub antibodies and non-immunized IgGs as control. Proteins were resolved on a 12% SDS–polyacrylamide gel and silver-stained (upper panel). The lower panel shows BER capacity of the corresponding immunoprecipitates using U/A- and AP/A substrates. (B) Western analysis of UNG2, PCNA, POL and XRCC1 in immunoprecipitates. (C) Reduction of UDG-activity in cell extracts by immunoprecipitation. ‘Pre-IP’ indicates UDG-activity prior to immunoprecipitation. The lower panel displays the corresponding western analysis of UNG proteins in cell extracts pre-IP and post-IP. (D) UDG activity of PU1sub- and non-immunized IgG beads. 1x, 7.5 μl input of beads; 2x, 15 μl input of beads.
Functional UNG2-ARC isolated using UNG2-specific antibodies contains proteins required for BER of uracil in DNA
Using whole cell extracts of HeLa cells and PU1sub-beads, we isolated UNG2-ARC that repaired U/G substrate, but such a complex could not be prepared using IgGs from pre-immune and non-immunized animals. Neither was repair of AP/G or nicked AP/G substrates observed when using unspecific IgGs for preparing complexes (Figure 4A). This demonstrates the requirement for specific antibodies and supports the view that UNG2-ARC is captured via UNG2. Furthermore, DNase I treatment of the extract did not reduce the yield of functional UNG2-ARC. Thus, the components of UNG2-ARC are not co-precipitated via common DNA molecules (Figure 4A).
Figure 4. Specificity in isolation of functional UNG2-ARC and western analysis of proteins present. (A) UNG2-ARC was prepared/attempted to be prepared using PU1sub coated beads, beads coated with non-immune IgG, or pre-immune IgG, which were all incubated with 400 μg HeLa whole cell extracts. The beads were incubated with BER assay mixture containing dCTP, and U/G, AP/G or AP-nick/G substrate, as indicated. In lane 2, extract was pretreated with 3 U DNase I at 30°C for 30 min prior to UNG2-ARC isolation. Purified DNA was digested for short-patch analysis. (B) Proteins from beads prepared as in (A) were mixed with denaturing loading buffer, heated at 70°C, separated by electrophoresis, blotted and visualized as described in Materials and Methods.
In general, results from western analysis supported the functional assays. We detected UNG2, APE1, PCNA, POL, POL? and XRCC1 in the UNG2-ARC (Figure 4B). We did not detect DNA ligase I possibly because of the properties of the antibody, since we only detected a weak band corresponding to DNA ligase I in whole cell extract using the same antibody. We did not investigate the possible presence of DNA ligase III. We also did not detect APE2, SMUG1, POL, POL, TDG or UNG1 in the UNG2-ARC, although they were all detectable in whole cell extracts (data not shown). The different intensities of the bands do not necessarily reflect the molar ratio of the proteins in the complex, but may merely indicate different properties of the antibodies used for western analysis. As control, we carried out similar experiments using pre-immune IgG linked to beads. No UNG2 was precipitated non-specifically (Figure 3B). The absence of detectable PCNA in the control suggests that PCNA is captured via interaction with immobilized UNG2, with which it is known to interact directly (22). Precipitation of POL was strongly reduced, but not completely absent with pre-immune IgG (Figure 4B). Pre-treatment of the extract with DNase I did not affect the capture of UNG2, PCNA or POL significantly, in agreement with the functional BER assays (Figure 4A).
We also found that proficient UNG2-ARC could not be prepared from an UNG–/– human lymphoblastoid cell line (36), strongly supporting the view that the BER complex is attached via UNG2 (Figure 5A). As an additional control, we successfully prepared proficient UNG2-ARC from peripheral blood lymphocytes, as well as cultured myeloma cells (data not shown). Using whole cell extract from hUNG–/– cells instead of UNG2-ARC, we detected repair of all three DNA lesions, indicating UNG2-independent BER in the extracts (Figure 5A). In agreement with this result, we could not detect any of the BER factors attached to beads coated with PU1sub when we attempted to isolate UNG2-ARC from UNG–/– lymphoblastoid cells (Figure 5B). Furthermore, UNG2 was not detectable in whole cell extracts from these cells, while PCNA, POL, POL? and XRCC1 were all identified (Figure 5B).
Figure 5. BER and western analysis with UNG–/– human lymphoblastoid cells. (A) Whole cell extract was prepared from UNG–/– human lymphoblastoid cells. Whole cell extract (50 μg protein) was used in BER assays with dCTP and substrates as indicated (lanes 4–6), or ‘UNG2-ARC’ was attempted to be prepared from whole cell extracts using PU1sub antibody, and used for BER assays with substrates as indicated (lanes 1–3). (B) Protein in whole cell extract (lane 2) or from immunoprecipitated ‘UNG2-ARC’ (IP) attempted to be prepared from whole cell extract using PU1sub (lane 1) were subjected to western blot analysis as in Figure 4B.
These experiments demonstrate that isolation of functional UNG2-ARC requires interaction between UNG2 and UNG2-specific antibodies and that complex formation is dependent on protein–protein interactions rather than protein–DNA interactions.
BER activity for uracil-containing DNA in UNG2-ARC and whole cell extracts has preference for U/G over U/A
To further examine the BER process in UNG2-ARC, we used a cccDNA substrate containing uracil opposite adenine (U/A) at the same position as in the U/G substrate. This eliminates possible differences related to sequence dependency of the repair process. As demonstrated in Figure 6, we consistently found that repair of U/G was better than repair of U/A, and was not a result of different quality of the substrate as such. Repair of U/A (upper panel) and U/G (lower panel) by UNG2-ARC was abolished by PU101, while a neutralizing anti-SMUG1 antibody (7) had no detectable inhibitory effect. This is consistent with our failure to detect SMUG1 in the complexes. Furthermore, in the presence of PU101, repair of U/A by whole cell extract after 60 min incubation was essentially undetectable (Figure 6, upper panel), and repair of U/G after 10 min was approximately 80% reduced (Figure 6, lower panel). This suggests that UNG2 is the major glycosylase responsible for repair of uracil-containing DNA in U/A pairs and U/G mispairs. However, whole cell extracts carried out a delayed, but significant repair of U/G after prolonged incubation in the presence of PU101 antibodies (Figure 6, lower panel). In whole cell extracts anti-SMUG1 antibodies alone had no significant effect on BER (Figure 5, lane 6), but in the presence of PU101, a small but further increase in inhibition by anti-SMUG1 antibodies was observed, when compared with PU101 alone (Figure 6, lane 8). These results indicate that UNG2 is the main, if not sole, enzyme for repair of U incorporated opposite of A, and the major enzyme for repair of U/G mispairs resulting from deamination of C. Furthermore, SMUG1 may have a back-up function, but is not the primary enzyme responsible for repair of U/G mispairs in this in vitro system. The failure of PU101 antibodies and anti-SMUG1 antibodies to completely inhibit BER when added together may indicate that the two other uracil-DNA glycosylases known, TDG and MBD4, as well as the newly identified DNA glycosylases NEIL1 and NEIL2 (40) may also be considered as potential candidates for the ‘delayed’ U/G repair. Although TDG has very low turnover of substrate alone, the rate is enhanced by APE1 (41), which is certainly present and active both in whole cell extract and in the complexes. Although unlikely because of the small patch size, we could not formally exclude DNA repair mechanisms other than BER in the whole cell extracts; e.g. nucleotide excision repair (NER) or mismatch repair (MMR). However, MMR repair was excluded as a significant source of repair in this system by using nuclear extracts prepared from MMR-deficient HCT-116 colorectal cancer cells (ATCC CCL-247) in the presence of both neutralizing antibodies, which again did not abolish repair completely (data not shown). Finally, these results indicate that UNG2 is the only uracil-DNA glycosylase present in UNG2-ARC.
Figure 6. Roles of UNG2 and SMUG1 in short-patch repair of U/A pairs and U/G mispairs. UNG2-ARC or whole cell extract of HeLa cells was incubated with BER assay mixture for 60 min with dTTP and U/A (upper panel), or with dCTP and U/G substrate (lower panel) in the absence or presence of anti-UNG antibody (PU101) or anti-SMUG1antibody (PSM1) or both, as indicated. The diagram represents results from four independent experiments where bars indicate SD.
Inhibition of UNG2 does not impair subsequent BER steps and recombinant SMUG1 partially restores BER of U/G by UNG2-ARC, but not U/A repair
To test whether inhibition of UNG2 affects subsequent BER steps carried out by UNG2-ARC, we assayed repair of U/G, AP/G and nicked-AP/G DNA substrates in the presence of neutralizing PU101 antibody. The antibodies completely inhibited repair of U/G, whereas AP/G and nicked AP/G substrates were effectively repaired (Figure 7A). Addition of 2 ng of recombinant SMUG1 enzyme (7) to PU101-inhibited UNG2-ARC, partially restored BER capacity for U/G and this restoration was inhibited by neutralizing SMUG1 antibodies, as expected. Next, we tested repair of U/A substrate under similar assay conditions. Neutralizing PU101-antibodies also inhibited repair of U/A completely, while repair of AP/A and nicked AP/A was not affected (Figure 7B). Contrary to experiments with U/G substrate, 2 ng recombinant SMUG1 enzyme did not restore BER of U/A. SMUG1 was previously shown to remove U from U/A pairs, although kcat/Km is 3.2-fold higher for U/G than for U/A in uracil release assays (7). These findings may in part explain the inability of SMUG1 to complement UNG2 in repair of U/A pairs.
Figure 7. Lack of functional coupling between the glycosylase step and the subsequent steps in short-patch repair. (A) UNG2-ARC was incubated with BER assay mixture with dCTP and U/G, AP/G or AP-nick/G as indicated, in the absence or presence of anti-UNG antibody (PU101) and/or anti-SMUG1 antibody (PSM1), and absence or presence of 2 ng recombinant SMUG1 (rec. SMUG1). (B) UNG2-ARC was incubated as in (A), but with dTTP and U/A, AP/A or AP-nick/A substrate.
APE1 is indispensable for BER activity and immunodepletion of XRCC1 reduces BER capacity of UNG2-ARC and the yield of ligated repair product
Mammalian cells have two AP-endonucleases, APE1 and APE2, that share homology with Escherichia coli exonuclease III (ExoIII). To test the relative contribution of these to the AP-endonuclease activity of UNG2-ARC, we conducted BER assays in the absence or presence of polyclonal anti-APE1 antibodies. We found that APE1 antibodies completely inhibited BER activity of UNG2-ARC (Figure 8A), indicating that APE1 is the only functional AP-endonuclease in UNG2-ARC. Further, we tested the functional role of XRCC1 in UNG2-ARC, and found that polyclonal antibodies to XRCC1 had no direct inhibitory effect in BER (Figure 8A). However, immunodepletion of XRCC1 resulted in markedly reduced BER, and a significant fraction of repaired products remained unligated after XRCC1 depletion (Figure 8B). These results are consistent with the presumed role of XRCC1 as scaffolding protein that interacts with other BER proteins, including POL?, DNA ligase III, and the fact that XRCC1 itself has no known catalytic activity.
Figure 8. Roles of APE1 and XRCC1 in BER. (A) UNG2-ARC was incubated with BER assay mixture in the absence or presence of APE1 and XRCC1 polyclonal antibodies as indicated. (B) Whole HeLa extracts were incubated with XRCC1-antibody linked beads (lanes 2 and 4) or non-immunized serum IgG-linked beads (lanes 1 and 3) for 4 h at 4°C. The beads were removed and the extracts were further incubated with PU1sub-linked beads. The UNG2-ARC thus captured was subsequently used in BER assay.
UNG2-ARC isolated from growth-arrested HaCaT cells display lower BER activity than UNG2-ARC isolated from exponentially growing cells
To examine BER activity of UNG2-ARC in relation to proliferative status of the cells, we isolated complexes from cell extracts prepared from exponentially growing cells (EC) and growth-arrested (AC) HaCaT cells. The cell cycle status of the cells was determined by FACS analysis (Figure 9A). Using the BER assay, we found a markedly lower BER activity (short- and long-patch) after 15 min in UNG2-ARC isolated from growth-arrested HaCaT cells compared with those prepared from exponentially growing cells (Figure 9B and C, lanes1 and 2, and diagram E). However, after 60 min incubation the BER process had reached equal levels of short-patch repair in the complexes (Figure 9B, lane 4 and 5, and diagram E), whereas long-patch repair of UNG2-ARC isolated from arrested cells was only 30% of that from growing cells (Figure 9C, lane5 and 6, and diagram E). This is consistent with the roles of several DNA replication factors in long-patch BER, implying close association of these DNA metabolizing systems. Furthermore, using the BER assay, we found similar patterns of repair in the corresponding whole cell extracts (data not shown). Addition of neutralizing anti-POL? antibodies to the BER mixture resulted in stronger inhibition of repair activity of UNG2-ARC prepared from the growth-arrested cells compared with that from growing cells (Figure 9B and C, compare lanes 4 and 5 with lanes 7 and 8, respectively, and diagram E). This indicates that POL? is the predominant repair DNA polymerase in the UNG2-ARC isolated from arrested cells. Adding 3-fold higher amounts of neutralizing anti-POL? antibodies to the repair reaction had no additional inhibitory effects (data not shown). In a previous study, inhibition of POL? was found to reduce long-patch by nearly 75% in cell extract, indicating involvement of POL? in this pathway (42). This agrees well with our results demonstrating reduction of long-patch BER when POL? activity was inhibited by neutralizing antibodies (Figure 9C, lanes 7 and 8). This indicates that either a fraction of long-patch repair in UNG2-ARC is initiated by POL? (10,43) or that POL? may synthesize repair patches longer than one nucleotide (11).
Figure 9. BER activity in proliferating and non-proliferating cells. UNG2-ARC was isolated from whole extract of exponentially growing HaCaT cells (EC) as well as from whole extract of high-density and growth-arrested HaCaT cells (AC) that did not replicate DNA. (A) Comparison of DNA content distributions as analyzed by flow cytofluorometry analysis of propidium iodide stained HaCaT cells. Grey histogram; exponentially growing proliferating cells, white histogram; growth-arrested non-proliferating cells. Fluorescence intensity is on the linear axis, while the vertical axis indicates the relative number of stained cells. UNG2-ARC was isolated from the extracts and used in BER assays with U/A substrate and dTTP isotope and incubated for indicated time periods in the absence (lanes 1–6), or presence (lanes 7–8) of neutralizing POL? antibodies. The repair products were purified and digested with restriction enzymes to release short-patch (B) and long-patch (C) repair products as well as total repair products (ligated/unligated products, BamHI/PstI) (D). Results calculated from two independent experiments, each in duplicate, for BER kinetics analysis of exponentially growing cells relative to growth-arrested HaCaT cells after 15 min incubation and in the presence and absence of anti-POL? antibodies after 60 min BER incubation (E).
The capacity of BER activity of cell extracts and UNG2-ARC is rate-limited not solely by the cellular content of UNG2
Base excision repair proteins has been suggested to be sequentially recruited to the site of repair, implying that repair complexes are generated in proportion with damage to DNA (44). Alternatively, the buildup of repair complexes such as UNG2-ARC could depend on the concentration of repair factors. For this purpose, we prepared extracts from ordinary HeLa cells and stably transfected HeLa cells overexpressing UNG2 (HTO-UNG2-45). We found 15-fold higher level of UDG enzymatic activity in induced cells compared with non-induced cells (data not shown). To examine uracil BER capacity in extracts and UNG2-ARC, we used U/A substrate because repair of this lesion is completely dependent on functional UNG, in contrast with U/G repair (Figure 6). The rate of uracil repair was increased 4-fold in cell extract prepared from induced cells compared with non-induced cells at 5 and 10 min incubation (Figure 10, lanes 1–6 and the panel below). The increase in BER capacity of UNG2-ARC isolated from UNG2-overexpressing cells was smaller (1.5- to 2.5-fold, lanes 7–10 and the panel below). The data from crude extracts indicate that UNG2 may be a rate-limiting factor, but since the BER rate increases 4-fold when UNG2 increases15-fold, it does not seem to be the sole rate-limiting factor. The results of BER analysis of UNG2-ARC indicate that formation of repair complexes could be independent of the presence of damage in DNA.
Figure 10. Increased cellular UNG2 protein enhances BER activity in whole cell extract and UNG2-ARC. Exponentially growing HeLa cells carrying a UNG2 expressing construct were either induced with 2 μg/ml of doxycycline for 48 h (+) or mock-induced (–) as control. UNG2-ARC was prepared by incubating 40 μl of PU1sub-linked beads with 100 μg of whole cell extract for each reaction. BER assays with 500 ng of U/A DNA substrate and dTTP as isotope were carried out using whole cell extracts (40 μg) (lanes 1–6) or UNG2-ARC (lanes 7–18) for the indicated incubation times. The panels below show the results calculated from two independent experiments.
DISCUSSION
Our work demonstrates the existence of a BER complex that is proficient in repair of uracil-containing DNA and western analysis confirmed that the factors known to be required for the process were present in the complex. Furthermore, the buildup of the complex is not dependent on the presence of uracil-damaged DNA. This indicates that at least one form of BER for uracil-containing DNA takes place in a preassembled multi-protein complex. Alternative models assuming sequential recruitment of enzymes to the site of repair have been suggested (45). Several observations, particularly the pattern of protein–protein interactions, support this model in which an incoming protein is recruited by one or more proteins present at the site of repair. After release of the base in the initial step, the DNA glycosylase may remain bound to the product DNA (AP-site) for which it has high affinity, and may shield it from damage (45). In some cases, DNA glycosylases have virtually no turnover (TDG), or low turnover (SMUG1), when present alone, but the turnover is enhanced several-fold by AP-endonuclease (7,41). The structure of APE1 bound to abasic DNA showed that APE1 uses a rigid, pre-formed surface to bend the DNA helix by 35° and retains its kinked DNA product efficiently (46). The kinked APE1–DNA complex is proposed to recruit POL? to sites of damage (46,47), where it binds to the nicked DNA, and induces a further bend to 90° (47). DNA kinking could also provide an effective means of promoting the directional and sequential exchange of factors at each step of the BER pathway (46). This recruitment model does not require the formation of a more stable BER complex, but is not incompatible with the preassembled BER complex model, where the substrate may be handed from one component of the complex to the other. One possibility is that these mechanisms operate in different contexts. Some components may be recruited as single proteins and others as small complexes, forming larger complexes with limited lifetime. Another possibility, in agreement with our results, is that the biochemistry of the individual steps operates just as suggested for the sequential recruitment model, but that this process simply takes place in a complex.
In a previous paper, a multi-protein BER complex from bovine testis containing POL?, DNA ligase I and uracil-DNA glycosylase was isolated by a POL? affinity method, and if proteins were present in equimolar concentrations, the complex had a cumulative mass of 196 kDa (20). This complex was captured in the absence of DNA by POL?-affinity chromatography. This argues against damage recognition by the glycosylase being a trigger for complex assembly. The fact that BER complexes can be isolated by two different methods supports the existence of such complexes in the cell. However, our work indicates that PCNA, POL, XRCC1, a DNA ligase and APE1 are also part of a BER complex, which must then be significantly larger than the complex reported previously. Possibly, these differences may be explained by the fact that we used lower concentrations of monovalent salt during isolation and washing of the complex. Since we did not detach the BER complex(es) from the beads, and did not examine them as complexes in solution, it is possible that UNG2 is present in complexes with different compositions. In NER, at least some repair factors are recruited sequentially to the site of damage after localized subnuclear exposure to ultraviolet light. Here the damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors (48). Thus, at best a fraction of NER proteins is present in preassembled repair complexes.
Based on experiments using isolated nuclei (22) and Ung-deficient mice (49), a role for UNG2 in removal of misincorporated uracil in U/A pairs has been established. However, the roles of UNG2, SMUG1 or other enzymes, in removal of uracil from U/G mispairs have remained unsettled (7,49). We find that UNG2-ARC and whole cell extracts are at least as effective in repair of U/G mispairs, as with U/A pairs. Previously, we defined kinetic properties of homogenous recombinant SMUG1 and UNG2 using U/A or U/G substrates in oligonucleotide cleavage assays. We concluded that these properties were consistent with a major role for UNG2 in repair of U/G mispairs, as well as U/A pairs. Complete BER in crude extracts of HeLa cells supported this view, in agreement with results presented here (7). However, a different paper concluded that murine SMUG1 has the major uracil-DNA glycosylase activity initiating repair on U/G mispairs, with UNG2 as a back-up (50). In these studies, the glycosylase step was examined using nuclear extracts in oligonucleotide cleavage studies, and low substrate concentrations were used to mimic the in vivo situation. The different conclusions reached in the latter study and the present study are not due to differences in the concentration of DNA-uracil, which are fairly equal in the two studies. It could, however, possibly be due to a much higher total DNA concentration but much lower relative lesion density used in our study as compared with study on murine extracts (50). The different results are, however, more probably explained by the use of different methods for preparation of extract, and by different kinetics due to assay conditions. It should also be noted that activities and kinetic properties of human UNG2 (7) and human and mouse SMUG1 (7,49) are dependent on concentrations of monovalent salts and Mg2+, as well as the presence or absence of APE1. Contrary to the kinetic properties originally reported for Xenopus SMUG1, and the erroneous entries of kinetic parameters for hUNG in this paper (51), kinetic parameters for human SMUG1 and UNG2 would be consistent with a major role for UNG2 and a backup function for SMUG1 in removal of uracil from DNA both in U/G mispairs and U/A pairs (7,52). Thus, the Km values of SMUG1 and UNG2 are 1.3 μM and 0.4 μM, respectively, whereas Kcat for UNG2 is 100-fold higher than that of SMUG1 and Kcat/Km some 300-fold higher than that of SMUG1 (7). Even if different assay conditions may modify the picture several-fold, the properties of SMUG1 does not make it a likely primary candidate in performing the important first step in BER of uracil-containing DNA. Our present data, both with BER complexes and whole cell extracts, supports this view. Furthermore, murine (53) and human UNG2 (36) have been demonstrated to be required for removal of uracil in U/G pairs resulting from deamination by AID in somatic hypermutation and class switch recombination in B-cells. The major role of SMUG1 is more likely to be in repair of oxidative base lesions, such as 5-hydroxymethyluracil (7,52,54) 5-hydroxyuracil and 5-formyluracil (52), where alternative repair enzymes are not identified, or are less efficient. Thus, we conclude that UNG2 is the major enzyme initiating BER of uracil-containing DNA both in a U/G and a U/A context. UNG2-ARC also proficiently repair abasic sites, but whether this type of complex represents the major type, completing repair initiated by glycosylases other than UNG2 remains to be examined.
Components of short- and long-patch BER have been identified and their roles studied both in vitro and in vivo as well as in reconstitution systems. However, mechanisms that determine the branching of BER pathway into short-patch and long-patch repair are not completely understood. The type of DNA damage was found to determine the mode of pathway (55). Thus, repair of hypoxantine (HX) and 1,N6-ethenoadenine (A) by the monofunctional alkyl-N-purine glycosylase (ANPG/MPG) was via both short- and long-patch BER, whereas repair of 8-oxo-G by the bifunctional hOOG1 protein was mainly via short-patch pathway (55). However, in a recent study on repair of 8-oxo-G, 55–80% repair patches were >1 nt in an in vivo system (56). However, the biochemistry of the repair pathway was not analyzed in this study, nor was possible impact of cell cycle status on the mode of repair. Modified 5'-dRp residues are refractory to the dRPase activity of POL? (57). Thus, repair DNA synthesis of such lesions could be initiated by POL? (58), but further removal of the 5'-dRp and completion of repair is thought to require a ‘switchover’ to a PCNA and POL/ dependent long-patch pathway (59). Although single nucleotide insertion is a dominant function of POL?, this DNA polymerase is able to replace several nucleotides containing a modified 5'-dRp, resulting in the generation of a flap oligonucleotide structure (11,60). The flap is cleaved by FEN1 (11), resulting in a nick in DNA which is subsequently sealed by a DNA ligase. A direct involvement of PCNA in BER outside sites of replication has not so far been demonstrated. However, a PCNA-independent long-patch BER outside sites of replication consisting of a core of four proteins; APE1, POL?, FEN1 and DNA ligase III may occur. Such long-patch repair might be the main mode of BER of modified AP-sites in resting cells. This model may be consistent with previous reports (11,42) as well as our data. The rate-limiting step in BER has previously been studied and suggested to be the removal of 2-deoxyribose 5-phosphate (dRp) when BER is initiated by a monofunctional glycosylase (e.g. UNG) (61), or the removal of 3'-blocking deoxyribose residue when a base damage is removed by a bifunctional DNA glycosylase (e.g. hOGG1) (62). Moreover, in a separate study, the joining of repair ends by a DNA ligase was suggested to be a rate-limiting step in BER (63,64). Although the rate of excision of uracil from DNA by UNG-proteins is highly sequence dependent (35), UNG2 and other UNG-proteins have very high catalytic turnover numbers (7,65). This suggests that glycosylase step is not the most likely rate-limiting step in UNG2-ARC and probably not in extracts. Previously, the excision of certain base lesions was also found to be a rate-limiting step in BER when a DNA glycosylase with low turnover activity removed the lesions (66). Apparently, the type and the position of a damaged base are additional factors in the kinetics of BER. In fact, we found that placing uracil opposite adenine at the neighboring nucleotide at the 5'-side of the U/A substrate used in our experiments (Figure 1) resulted in a significant reduction of rate of repair (data not shown).
In conclusion, we immunoprecipitated BER complexes that carry out short-patch and long-patch BER for uracil-containing DNA, using antibodies specific for the N-terminal non-catalytic part of UNG2. Short-patch repair is the predominant mode both in extracts and in BER complexes. Furthermore, UNG2 appears to be the major glycosylase responsible for repair of U/G mispairs and possibly sole enzyme for repair of U/A pairs in human cells. On the basis of the results presented here, we suggest that at least a significant fraction of BER proteins for repair of uracil-containing DNA, and possibly for other BER substrates as well, are present in preassembled repairosome multiprotein BER complexes. These may be dynamic of nature.
ACKNOWLEDGEMENTS
This work was supported by The Research Council of Norway, The Norwegian Cancer Society, The Cancer Fund at St Olav's Hospital, Trondheim, and The Svanhild and Arne Must Fund for Medical Research, INSERM and the European Economic Community (contract number QLG1-CT-2001-01536- IMPAD). J.P.-D. is a recipient of a Marie Curie fellowship from the European Community programme IHP-MCIF-99-1 under contract number HPMF-CT-2001-01290.
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