Transcription influences the types of deletion and expansion products
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《核酸研究医学期刊》
Institute of Biosciences and Technology, Center for Genome Research, Texas A&M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Blvd, Houston, TX 77030, USA
* To whom correspondence should be addressed. Tel: +1 713 677 7651; Fax: +1 713 677 7689; Email: rwells@ibt.tamu.edu
ABSTRACT
The genetic instability of (GAC?GTC)n (where n = 6–74) was investigated in an Escherichia coli-based plasmid system. Prior work implicated the instability of a (GAC?GTC)5 tract in the cartilage oligomeric matrix protein (COMP) gene to the 4, 6 or 7mers in the etiology of pseudoachondroplasia and multiple epiphyseal dysplasia. The effects of triplet repeat length and orientation were studied after multiple replication cycles in vivo. A transcribed plasmid containing (GAC?GTC)49 repeats led to large deletions (>3 repeats) after propagation in E.coli; however, if transcription was silenced by the LacIQ repressor, small expansions and deletions (<3 repeats) predominated the mutation spectra. In contrast, propagation of similar length but opposing orientation (GTC?GAC)53 containing plasmid led to small instabilities that were unaffected by the repression of transcription. Thus, by inhibiting transcription, the genetic instability of (GAC?GTC)49 repeats did not significantly differ from the opposing orientation, (GTC?GAC)53. We postulate that small instabilities of GAC?GTC repeats are achieved through replicative slippage, whereas large deletion events are found when GAC?GTC repeats are transcribed. Herein, we report the first genetic study on GAC?GTC repeat instability describing two types of mutational patterns that can be partitioned by transcription modulation. Along with prior biophysical data, these results lay the initial groundwork for understanding the genetic processes responsible for triplet repeat mutations in the COMP gene.
INTRODUCTION
The etiologies of approximately 14 inherited neurological disorders are due to the genetic instabilities of triplet repeat sequences (TRSs) . Three of the ten possible triplet repeats have been associated with the widely studied diseases such as myotonic dystrophy, Huntington's disease, spinocerebellar ataxias, fragile X syndrome and Friedreich's ataxia, which involve expansions of CTG?CAG, CGG?CCG and GAA?TTC repeats in the relevant genes (1,2). A fourth triplet repeat (GAC?GTC) was identified (3) in the cartilage oligomeric matrix protein (COMP) gene, which exhibited small expansions and deletions (1 or 2 repeats) from the parental 5mer. A single trinucleotide expansion leads to multiple epiphyseal dysplasia (MED), whereas two triplet expansions (or one repeat deletion) lead to pseudoachondroplasia (PSACH). MED and PSACH are clinically characterized by joint pain, short stature, early-onset osteoarthritis and limb dwarfism (3). Since a single TRS deletion or expansion is sufficient for disease onset, unstable GAC?GTC repeats contrast with other TRS-induced diseases that require larger or even massive TRS expansions to cause disease (1,2). Previously, GAC?GTC repeats were shown to be genetically unstable in a plasmid-based system (4), several years before this association was made to a disease state. In addition, comparative biophysical data (5–8) on synthetic oligonucleotides of GAC to CAG repeats as well as GTC to CTG repeats revealed similar hairpin folding properties in vitro. Thus, since the genetic instability of the CAG?CTG sequence has been intensely studied , we intended to evaluate whether these results would be applicable to GAC?GTC repeats.
Replication, recombination and DNA-repair-based mechanisms have been investigated to explain the genetic expansions of TRS in bacteria, yeast, cell culture and mammalian systems (1,9,10). Repeat instability is dependent on several factors, including the length and type of the TRS, the orientation of the repeats with respect to the direction of replication, the number of replication cycles and the genetic background of the host cells (9). These factors are not mutually exclusive. In addition, transcription was shown to cause increased levels of deletions in CAG?CTG repeats (11–13). One model for this behavior envisioned the non-transcribed strand adopting folded-back secondary structures in long CAG?CTG tracts; after several replication cycles, an increased frequency of deletions was observed in an orientation-dependent manner (12).
Herein, we investigated the mechanisms influencing instability in a plasmid-based system harboring various lengths of GAC?GTC repeats (6–74 repeats) to determine the types and amounts of expansions and deletions during multiple replication cycles in Escherichia coli. In addition, induction or repression of transcription through these repeats revealed that two types of mutational patterns, small and large length changes, are partitioned in an orientation- and length-dependent manner.
MATERIALS AND METHODS
Plasmids
The plasmids used in this study are listed in Table 1. Some plasmids containing GTC?GAC repeat tracts were constructed and characterized previously (14); pRW3413 (14) was used herein. Other GTC?GAC containing plasmids that were prepared and partially characterized (K. Ohshima and R. D. Wells, unpublished data) were sequenced; these included pRW3132, pRW3131, pRW3815 and pRW3820. Plasmids pRW4606, pRW4607 and pRW4608 are derivatives of pRW3413, pRW3815 and pRW3820, respectively. In order to clone the repeats in the opposing orientation, the inserts from pRW3413, pRW3820 and pRW3815 were released by HindIII and SacI digestion. Each insert from pRW3413, pRW3820 and pRW3815 was separated on a 7% acrylamide gel, stained with ethidium bromide and the TRS-containing bands were excised. The eluted DNAs from the excised bands were purified by phenol–chloroform extraction and precipitated with ethanol (15). The purified inserts were ligated to the corresponding linearized vector (pUC18 or pUC19) at approximately a 1:10 molar ratio with 20 U of T4 DNA ligase (United States Biochemical Corp.). The reactions were carried out at 16°C for 12 h. The ligated products were ethanol precipitated and transformed into E.coli HB101 by electroporation, and the transformed cells were plated on Luria–Bertani (LB) agar plates containing 50 μg/ml ampicillin (15). While cloning the inserts into the pUC vectors for the opposing orientation, most TRS-containing inserts were deleted by a few repeats.
Table 1. Plasmids used in this study
In order to attempt to obtain plasmids containing undeleted TRS inserts, each plasmid (Table 1) containing GAC?GTC and GTC?GAC repeats was electrophoresized on a 1% agarose gel. The corresponding band of supercoiled DNA was excised and eluted for phenol–chloroform extraction. After purification, the DNA was transformed into E.coli HB101, and the cells were spread onto an agar plate with ampicillin (50 μg/ml). Individual colonies were grown overnight at 37°C in liquid LB medium. DNAs from individual colonies were isolated by a Wizard Plus Minipreps DNA Purification System (Promega) and then analyzed by restriction mapping and sequencing. The length of the repeat from each plasmid (Table 1) was determined by digestion with HindIII and SacI to release the TRS-containing insert, and the fragments were radiolabeled with dATP (15) and resolved on a 7% native acrylamide gel. Dideoxy sequencing of both strands with a Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (United States Biochemical Corp.) was utilized for determining the sequence and orientation of the TRS-containing plasmid. The sequencing reactions were carried out according to the manufacturer's recommendations. The following primers were utilized: M13/pUC sequencing primer 5'd(GTAAAACGACGGCCAGT)3' (#S1211S New England Biolabs, Inc.) and M13/pUC reverse sequencing primer 5'd(AACAGCTATGACCATG)3' (#S1201S New England Biolabs, Inc.). The products of the sequencing reactions were analyzed on 6% acrylamide Long Ranger gels (FMC BioProducts) containing 8 M urea in glycerol tolerant buffer (United States Biochemical Corp.). The gels were dried and exposed to X-ray film.
Recultivation assay and product analyses
Each plasmid containing uninterrupted GAC?GTC and GTC?GAC repeat tracts were transformed into E.coli AB1157 . In order to obtain multiple recultivation experiments for each plasmid, three or more aliquots of each transformation mixture were grown overnight at 37°C in 10 ml liquid LB plus ampicillin (50 μg/ml) at a shaking rate of 250 r.p.m.; alternatively, a number of repeated transformations were performed for each plasmid. Each 10 ml liquid culture was designated as growth cycle 1. An aliquot of each growth cycle 1 culture was diluted 107-fold for inoculation into in a new 10 ml liquid LB plus ampicillin and grown overnight under the same conditions as the first inoculation. Recultivation of these cultures was repeated through four growth cycles. In addition, to determine the transformation efficiency, an additional aliquot from each transformation mixture was plated onto a 1.5% agar plate with ampicillin (50 μg/ml). Approximately 100–1000 transformants were obtained for each transformation mixture. After each growth cycle, the populations of cells were harvested and plasmid DNA was isolated. DNA from cultures (growth cycles 1–4) was digested with HindIII and SacI to release the repeat-containing inserts which were then radiolabeled with dATP for analyses by 7% native gel electrophoresis at 37°C.
For the experiments with inhibited transcription, the plasmid pIQ-kan was transformed into the E.coli AB1157 host strain. The pIQ-kan plasmid expresses the repressor for the lacZ promoter thereby inhibiting transcription of the lacZ gene. The TRS were cloned downstream of the lacZ promoter and therefore transcription through the TRS tract was also inhibited. In the presence of the repressor, the recultivation assay was carried out as described above with the addition of kanamycin (100 μg/ml) selection for the pIQ-kan plasmid. When required, isopropyl-?-D-thiogalactopyranoside (IPTG) of 1 mM was included in the growth medium for the induction of transcription. Replication and transcription through the repeat from the lacZ promoter for both pUC18 and pUC19 are co-directional (15).
After electrophoresis of the radiolabeled molecules from a recultivation experiment, the gel was exposed to a PhosphorImager screen (Molecular Dynamics). The gel was scanned and the areas under each peak were determined by using the Image Quant program (Molecular Dynamics).
RESULTS
The effect of repeat tract length on the instability of (GAC?GTC)n and (GTC?GAC)n in transcribed plasmids
The genetic stability of CAG?CTG, CGG?CCG and GAA?TTC repeat-containing constructs is dependent on the length of the repeat tracts (16–23). In order to determine the effect of length on the stability of (GAC?GTC)n and (GTC?GAC)n, the appropriate plasmids (Table 1) (where n ranged from 6 to 74) were propagated in a recultivation assay (18,24). The (GAC?GTC)n and (GTC?GAC)n sequences were cloned into the polylinker of pUC plasmids and are located 400 bp from the origin of replication for both pUC18 and pUC19. The (GAC?GTC)n and (GTC?GAC)n tracts, located in the lacZ gene which is under the control of the lacZ promoter, were transcribed. For our considerations herein, large deletion or large expansion events obtained during a recultivation assay were designated as >3 repeats and small deletion or small expansion events were designated as <3 repeats. After each growth cycle in a recultivation assay, plasmid DNAs were analyzed by HindIII–SacI digestion and the TRS-containing inserts were radiolabeled and resolved by native gel electrophoresis as described under Materials and Methods.
pRW3132 and pRW3131 were propagated in E.coli AB1157 for four growth cycles. Small deletion and expansion events were observed. However, the amounts of the instabilities were small (<10%) (data not shown). Thus, in order to enhance the instability in our genetic assay, we lengthened the TRS tract to evaluate the molecular mechanisms for GAC?GTC instability. pRW4606 and pRW3413 containing longer TRS tracts were propagated as described above. Figure 1A demonstrates that increasing growth cycles cause small expansions and deletions of the plasmids harboring (GAC?GTC)27 and (GTC?GAC)30 while the progenitor length of both molecules was maintained. After the recultivation study, DNA sequencing and restriction analyses of the products (Materials and Methods) revealed that both plasmids had deleted and expanded products with repeat lengths that were close to the progenitor repeat length. For example, the small deletion and expansion products consisted of lengths 27, 24, 22, 23, 17 and 15 repeats for pRW4606 and39, 36, 33, 31, 30, 28, 25, 24 and 23 repeats for pRW3413. These products were stable when propagated through subsequent growth cycles. Hence, these data show that expansions and deletions changed by only small increments in transcribed pRW4606 and pRW3413.
Figure 1. Analyses of in vivo genetic instabilities of (GAC?GTC)n and (GTC?GAC)n repeats. The results of a representative recultivation experiment through four growth cycles (indicated as 1–4) in E.coli AB1157 are shown; the plasmid DNA products were digested with HindIII and SacI, and the fragments, radiolabeled with dATP, were separated on a native 7% acrylamide gel as described under Materials and Methods. The arrowheads indicate the full-length fragments (no expansions or deletions) for each parental plasmid. The products of expansions and deletions are shown as a function of the growth cycles of plasmids containing (A) (GAC?GTC)27 and (GTC?GAC)30 repeats (B) (GAC?GTC)49 and (GTC?GAC)53 repeats and (C) (GAC?GTC)74 and (GTC?GAC)69 repeats.
pRW4608 and pRW3820, which are similar in length, with 20 more repeats than the DNAs described above, were also propagated in recultivation assays. The restriction analyses shown in Figure 1B demonstrate a loss of the full-length progenitor fragment with increasing growth cycles for pRW4608 and retention of the full-length fragment with increasing growth cycles for pRW3820. Furthermore, the pattern of genetic instabilities observed for the two molecules differ. For pRW4608, with a length of 49 repeats, the progenitor fragment is deleted entirely after the second growth cycle to give deletion products that are predominately large deletion events. The major instability events for pRW4608 had lengths of 61, 51, 49, 48, 46, 41, 32, 26, 19 and 14. Most of the length changes were >3 repeats. Alternatively, for pRW3820 with 53 repeats in the opposing orientation, the progenitor fragment was retained at its full length even after four growth cycles while generating small deletion and expansion events. The expansion and deletion products from pRW3820 consisted of lengths 63 and 59, all integral units from 57–47, 42, 40, 38, 34, 31 and all integral units from 30–19. Thus, the n was <3 for each member of this family of products. Hence, in actively transcribed plasmids, large deletion events predominated for pRW4608 and small expansion and deletion events predominated for pRW3820. From these data, the orientation of the insert was the major influencing factor in determining the type of instabilities obtained from pRW4608 and pRW3820.
Earlier studies (17,25–31) explained that a possible mechanism for the effect of insert orientation of CTG?CAG versus CAG?CTG repeats was due to the higher propensity of long CTG repeats to form a more stable hairpin structure than long CAG repeats on the lagging strand template. Thus, during replicative events, the single-stranded state of the lagging strand template allowed for preferential hairpin formation of CTG repeats than CAG repeats that could be consequently bypassed by DNA polymerase and subsequently lead to greater instability of CTG repeats than CAG repeats. Similar to CTG and CAG repeating sequences, we found that the mutational patterns were strongly dependent on insert orientation. For example, when the intensities of each band from Figure 1B was plotted as a function of migration distance (plot not shown), the peak corresponding to the progenitor full-length fragment containing (GAC?GTC)49 repeats diminished to baseline by growth cycle 3 and peak intensities corresponding to the large deletion products gained intensity with increasing growth cycles. However, in the opposing orientation, the intensity of the corresponding peak for pRW3820 was not only sustained throughout the four growth cycles, but the area of the base of the full-length peak broadened, indicating that small expansion and deletion events in the population were progressively accumulating near the full-length peak with successive growth cycles. Thus, pRW3820 containing (GTC?GAC)53 displayed the capacity to expand and delete by 1–3 repeats indicative of triplet repeat slippage during DNA replication, whereas the insert (GAC?GTC)49, more capable of hairpin formation, could render large deletion events due to possible hairpin bypass on the lagging strand template during DNA synthesis.
Plasmids with longer repeat tracts, (GAC?GTC)74 and (GTC?GAC)69, were also propagated for determining the extent of expansions and deletions. For both plasmids, the original full-length fragment from pRW3815 and pRW4607 were deleted entirely after the second growth cycle while generating large expansion and deletion products (Figure 1C). The products contained 77, 74, 71, 63, 55, 44, 38, 36, 35, 34, 22, 20, 18 and 15 repeats for pRW3815 and 69, 68, 64, 62, 44, 40, 34, 28, 23, 22, 20 and 16 repeats for pR4607.
In summary, these data show that GAC?GTC and GTC?GAC repeats are unstable in transcribed plasmids. Smaller length repeats (30 repeats) deleted and expanded by small increments whereas longer repeat lengths (70 repeats) deleted as large deletions. A length threshold was observed in the types of instabilities obtained after four growth cycles for pRW4608 and pRW3820 with 50 repeats. The mechanisms of the large instabilities from pRW4608 and the small instabilities from pRW3820 are further explored below by inhibiting transcription of the lacZ gene.
Quantitation of instability products from (GAC?GTC)n and (GTC?GAC)n
Figure 2 shows the percentages of expansions, deletions and full-length retentions of GAC?GTC and GTC?GAC tracts averaged from three independent recultivation experiments. Inserts in the GAC?GTC orientation with lengths of 27, 49 and 74 demonstrate that the percentage of the full-length repeat decreases as the length of the repeat sequence increases. In this orientation, expansion events were infrequent, but the percentage of deletions dramatically increased as the tracts lengthened from 27 to 74 repeats. By the third growth cycle for length 49, deletions were essentially the only type of mutation observed.
Figure 2. The amounts of products formed from (GAC?GTC)n and (GTC?GAC)n repeats as a function of growth cycles. The extents of genetic instabilities were determined by quantitating the areas of all peaks in each lane as described in Materials and Methods. The averages from three independent recultivation experiments (including experiments from Figure 1) of percentage expansions (filled bars), deletions (gray bars) and original length fragments (crosshatched bars) are plotted against the number of growth cycles (indicated as 1–4). The length and orientation of the inserts are denoted above each plot.
In the orientation GTC?GAC (Figure 2, right-hand side), the percentage of full-length progenitor fragment was sustained for pRW3413 containing length 30 and for pRW3820 containing length 53, but not for pRW3815 containing length 69. For length 30, the full-length fragment only diminishes by 18% from the initial growth cycle to the end of the fourth growth cycle whereas in the other orientation for pRW4606 containing length 27, the percentage is reduced by 74%. In addition, expansion events for pRW3413 with 30 repeats constituted 7% of the genetic instability that was sustained throughout the four growth cycles. With the same insert orientation but with the longer repeat, pRW3820 containing (GTC?GAC)53, the full-length fragment was reduced by 44% after four growth cycles and the expansion events constituted 10% of the overall percentage. As the length of the repeat array increases, for plasmids with similar length inserts , there is a loss of the full-length fragment by the third growth cycle from 37 to 0% and from 41 to 0%, respectively; thus, the effect of orientation was overcome by length.
Hence, these data show that the instabilities of the GAC?GTC and GTC?GAC repeats are dependent on the length of the tract. The rapid loss through the growth cycles of the full-length fragment in the GAC?GTC orientation compared to the GTC?GAC orientation indicates that GAC?GTC repeats deleted more readily than GTC?GTC repeats. In addition, the TRS mutations in the GTC?GAC orientation exhibited a significant percentage of expansions in addition to deletions.
Repression of transcription influences the types of genetic instabilities in (GAC?GTC)49 and (GTC?GAC)53 tracts
Prior investigations have shown that transcription through repetitive DNA increases the occurrence of deletions in bacteria (11–13,32,33) and in yeast (34). Two types of genetic instability patterns described as large deletion and expansion and small deletion and expansion events had been described previously in plasmids containing CTG?CAG repeats (24,35–37). Similarly, two different mutational patterns appeared in the recultivation of plasmids containing (GAC?GTC)49 and (GTC?GAC)53 repeats when transcription and replication were coupled (see above).
In order to determine the genetic instabilities arising from DNA replication-mediated events in GAC?GTC and GTC?GAC repeats, transcription was silenced. Transforming the pIQ-kan plasmid that synthesizes the LacIQ repressor into the E.coli AB1157 host inhibited transcription. The LacIQ repressor inhibits transcription from the lacZ promoter. Conversely, the addition of IPTG, a gratuitous inducer, stimulates transcription (32). After electroporation of the pIQ-kan plasmid into E.coli AB1157, the cells were additionally transformed with either (GAC?GTC)49 or (GTC?GAC)53 containing plasmids. The cultures were allowed to pass through four growth cycles, and the cells were harvested at approximately every 20 generations (one growth cycle) as described above.
Figure 3A shows the results of restriction analyses of pRW4608 and pRW3820 during four growth cycles. Large deletion events were dramatically reduced compared to data in the presence of basal transcription (Figure 1B) during the recultivation of pRW4608 in the presence of LacIQ repressor; the products consisted of small expansions and deletions as the predominant types of mutagenic events. pRW4608 was subcultured for eight generations to test whether the starting fragment length was also deleted; Figure 3 shows that the progenitor length was sustained as the predominant product (data from growth cycles 5–8 not shown). In contrast, the recultivation of pRW3820 under the same conditions did not show a significant alteration in the small deletion and expansion pattern as observed previously in the presence of transcription (Figure 1B), thus indicating that the silencing of transcription did not influence the instability of pRW3820. The percentages of the progenitor full-length fragment are diminished by only 8% for pRW4608, whereas pRW3820 was deleted by 31% by the end of four growth cycles (Figure 4A). Thus, large deletions in (GAC?GTC)49 are associated with transcription.
Figure 3. The effects of transcription silencing on the genetic instabilities of (GAC?GTC)49 and (GTC?GAC)53 repeats. E.coli AB1157 harboring pIQ-kan was transformed with the supercoiled monomers of the designated plasmids. The instability of the (GAC?GTC)49 and (GTC?GAC)53 inserts were analyzed as described in the legend to Figure 1 and in Materials and Methods. The growth cycles are indicated as 1–4. The full-length progenitor fragments of the each parental plasmid are indicated by an arrowhead. (A) The results from a representative recultivation experiment of pRW4608 and pRW3820 that were carried out in the presence of LacIQ repressor for determining the extent of genetic instabilities derived from replication-mediated events while lacZ gene transcription was inhibited. (B) The results from a recultivation experiment with pRW4608 and pRW3820 as a function of four growth cycles in the presence LacIQ repressor and IPTG.
Figure 4. The effects of transcription silencing on the percentage of genetic instabilities of (GAC?GTC)49 and (GTC?GAC)53 repeats. Instability studies similar to the data in Figure 3 were quantitated as described in Materials and Methods. (A) The extent of instabilities of pRW4608 and pRW3820 in E.coli AB1157 in the presence of the LacIQ repressor is expressed by percentages of expansions (filled bars), deletions (gray bars) and original full-length material (crosshatched bars) and plotted against multiple growth cycles (indicated as 1–4). (B) The percentages of genetic instabilities of pRW4608 and pRW3820 when transcription is restored by the addition of IPTG and LacIQ repressor. The percentages are averages of three individual experiments including Figure 3.
To confirm whether large deletions are caused by transcription-mediated events in the TRS of pRW4608, IPTG was used to overcome repression by the LacIQ repressor and thus induce transcription. As stated above, recultivations of pRW4608 and pRW3820 were carried out in parallel with the addition of IPTG in the growth medium for four growth cycles. As expected (Figure 3B), large deletion events were found during the four growth cycles of pRW4608 causing 5-fold destabilization of the progenitor full-length fragment (compared with data in Figure 3A). However, in addition to the large deletion events observed for pRW4608, the progenitor full-length fragment was also sustained throughout the four growth cycles with the addition of IPTG. The presence of both types of instabilities was probably due to either greater amounts of repressor molecules than IPTG molecules in certain cells, or, alternatively, the repressor molecules may have greater binding affinity to the lacZ promoter than to IPTG under these growth conditions. In support of the latter case, in a separate control experiment (data not shown), we observed that large deletions were not inducible when IPTG was introduced late (in growth cycles 5–8) in the presence of repressor during the recultivation of pRW4608. Thus, we conclude that LacIQ repressor is an effective inhibitor of large deletions despite the high amounts of IPTG molecules. In the opposing orientation, the IPTG induction of transcription did not affect the small expansion and deletion events which were observed previously with pRW3820 in Figure 3A (right-hand side).
Repression of transcription influences the genetic instabilities of TRS longer tracts
We hypothesize that large deletion events arise through a transcription-mediated mechanism involving DNA loop structures, whereas small expansion and deletion events are the result of a TRS slippage during DNA replication. To evaluate our hypothesis, the propagation of plasmids containing the longer lengths of (GAC?GTC)74 and (GTC?GAC)69 repeats should result in small deletion and expansion mutations if transcription were silenced rather than giving large deletion events as previously shown in Figure 1C. To test this hypothesis, pRW3815 and pRW4607, which harbor longer repeat lengths, were also grown for four growth cycles in the presence of the LacIQ repressor (Figure 5A). With transcription blockage (Figure 5B), both molecules sustained 30 and 34%, respectively, of the progenitor length fragment by the end of four growth cycles. In addition, small instabilities were observed where the full-length fragment was altered to lengths of all integral units from 73–67, 63, 60, 56, 53, 51, 47, 44, 42, 41, 39, 36, 33, 29, 26, 24, 21 from plasmids containing (GAC?GTC)74 repeats and to lengths of all integral units from 68–48, 46–43, 40, 38, 34, 30, 29, and 25, 24, 23 from plasmids containing (GTC?GAC)69 repeats. The majority of the length changes were <3 repeat units indicative of slippage during DNA replication. Hence, these results show that the inhibition of transcription not only stabilizes the parental plasmid even in longer TRS arrays but that transcription through the repeats affects the types of genetic instabilities that include large deletions when transcription is turned on to small deletions and expansions when transcription is turned off.
Figure 5. The effects of transcription silencing on the longer repeat tracts of (GAC?GTC)74 and (GTC?GAC)69. E.coli AB1157 harboring pIQ-kan was transformed with pRW3815 and pRW4607 for determining the influence of transcription through longer TRS repeats. (A) Through four recultivations, the DNAs were isolated and the TRS-containing inserts were released with HindIII and SacI digestion. The figure shows a 7% native gel electropherogram of the products through four growth cycles (indicated as 1–4) when transcription was inhibited by the LacIQ repressor. Arrowheads mark the full-length fragments derived from the parental plasmids. (B) The percentage of genetic instability is displayed for pRW3815 and pRW4607 through the four growth cycles. The expansions (filled bars), deletions (gray bars) and original progenitor full-length fragments (crosshatched bars) are plotted against the number of growth cycles. The percentages are averages of three individual experiments including the data in (A).
DISCUSSION
Two types of expansion and deletion products were partitioned by modulating transcription through GAC?GTC repeats in an E.coli model system. Small expansions and deletions in descending amounts relative to the full-length tract were observed for TRS lengths of <30 repeats and for (GTC?GAC)53 repeats under basal transcription conditions. In the opposing orientation, (GAC?GTC)49, the pattern of instabilities was different; large deletion products predominated in the population while the progenitor full-length tract was entirely deleted. However, if transcription was inhibited, the products were small expansions and deletions with the maintenance of the progenitor full-length tract. Interestingly, induction or inhibition of transcription through the (GTC?GAC)53 repeats did not alter the pattern or extent of small expansions and deletions indicating a threshold length in the type of instabilities obtained after successive growth cycles. Furthermore, the longer TRS lengths (>69 repeats) also generated large deletion products in both insert orientations but products could also be altered by inhibition of transcription. Thus, transcription through the repeats promotes large deletion products in long repeat tracts in an orientation-dependent manner; in contrast, inhibition of transcription through the repeats led to enhancement of small expansions and deletions independent of insert orientation.
Replication-based instability of (GAC?GTC)n and (GTC?GAC)n
Figure 6 shows a model to explain the occurrence of small instabilities; the length changes are proposed to occur during the discontinuous synthesis of the Okazaki fragments from the lagging strand template rather than during the continuous synthesis of the leading strand. During Okazaki fragment processing, the single-strand state of the DNA facilitates the formation of slipped structures (38,39). We propose that during DNA replication, the original length, N, would be conserved on the leading strand template , while misalignment of the repeat on the lagging strand template , would cause length changes. In the latter case, DNA polymerase would bypass the looped-out repeats from the lagging strand template that could potentially be carried through to the next round of replication . Thus, the new leading strand would have a deletion of one repeat from the original length and the new lagging strand would retain the progenitor length (N).
Figure 6. Model for DNA slippage during replication of GAC?GTC and GTC?GAC repeats when transcription is silenced. During DNA replication, the lagging strand synthesis allows for single-stranded regions to misalign, either on the lagging strand template (A) or on the lagging nascent strand (B) in the regions of the repeating tract. (A) While in (a), the leading strand template (top strand of fork) retains the progenitor length (N), in (b) DNA slippage occurs by one repeat on the lagging strand. The DNA polymerase complex is proposed to bypass the looped-out repeat on the lagging strand template (17). (c) In the subsequent replication cycle, the new leading strand template has a deletion of 1 repeat (N – 1) whereas the new lagging strand template retains the progenitor repeat length (N). (B) (a) is the same as described for (A). (d) If the slippage of 1 repeat occurs on the nascent lagging strand, (e) then the newly synthesized top strand in the subsequent replication cycle will have an expansion of one repeat (N + 1) while the lower lagging strand template retains the progenitor length (N) (17,24).
Alternatively, if synthesis in excess of one repeat unit (expansions) occurs by misalignment on the lagging nascent strand , then in the subsequent replication event the new leading strand would consist of an expansion (N + 1) and the new lagging strand would conserve the progenitor full-length fragment (N). Our data show that the progenitor full-length fragment was sustained throughout multiple growth cycles as the predominant product while accumulating small expansion and deletion products of descending lengths and intensities, particularly when the repeats were not transcribed (Figure 3A). Thus, this model would explain the probability of maintaining the full-length fragment in higher amounts than the expanded and deleted products.
Transcription-associated mutations in the repeats
Transcription into tracts of the myotonic dystrophy CTG?CAG repeats enhanced the instability of the repeats, especially deletions (11,13). We previously proposed a model that envisioned transcription-induced local positive and negative superhelical domains that could facilitate hairpin formation of the CTG?CAG repeats (12) on the non-transcribed strand. Figure 7 extends this model with the consideration of the effect of insert orientation when transcription occurs through similar length repeats (left-hand side), resulting in two types of mutational patterns. After the DNA duplex melting by RNA polymerase (40) and resulting negative supercoil unwinding, the DNA is unwound (41,42). The transient single-stranded state of the non-transcribed strand containing GTC repeats enables quasi-stable hairpin formation. After passage of the RNA polymerase complex and during DNA re-annealing, any hairpins on the non-transcribed strand may generate several unpaired bases on the complementary strand. Therefore, these unpaired bases may transiently remain in an unstable looped-out structure. These loci may be substrates for mismatch repair or NER . Prior studies with the E.coli LacI (44,45) and human hprt genes (46) showed that the unpaired non-transcribed DNA strand had a greater propensity for mutations than the transcribed strand. Furthermore, the non-transcribed strand was repaired 4 times slower than the transcribed strand in the human p53 gene (47). Thus, the non-transcribed strand may have a greater propensity to be mutated than the template strand due to the transient single-stranded state.
Figure 7. Model for the involvement of transcription in the instabilities of GAC?GTC and GTC?GAC repeats of an intermediate length. RNA polymerase causes local duplex melting at the TRS tract for the initiation of transcription (40). Local positive and negative superhelical domains aid the progression of the RNA polymerase through the duplex DNA (41,50). While the transcribed GAC strand (left panel) complexes with the newly formed mRNA, the GTC strand which is not transcribed can readily form a hairpin. Re-annealing of the duplex DNA after transcription termination allows for the remaining unpaired bases on the GAC strand to loop out. The hairpin or looped-out structures are potential substrates for DNA repair proteins such as those that are involved in MMR (43), NER (51,52) or the SOS response (43). It is proposed that the structures are excised, DNA polymerase fills-in the gaps and the nicks are sealed by ligase. The removal of several bases due to their engagement as a hairpin or looped-out structure leads to large deletions on both strands of the DNA. In the other TRS orientation (right panel), when the GTC strand pairs with the mRNA, the GAC strand becomes unpaired; however, the GAC strand at an intermediate length does not form a hairpin structure as readily as the GTC unpaired strand (left panel). Therefore, transcription through intermediate-length GTC?GAC repeats does not stimulate genetic instabilities.
In previous studies (11,12), when CTG?CAG repeats were propagated in E.coli HB101, the main determinant for the loss of progenitor full-length tract was the increased growth rates of bacterial cells harboring deleted products. Similarly, we found that transcription through (GAC?GTC)49 led to the complete loss of the progenitor full-length tract with increasing amounts of large deletions. However, this interpretation was not plausible when transcription was inhibited through the repeat. Instead, we observed that the progenitor full-length tract was present in higher amounts than the expanded and deleted products. Furthermore, the small length changes never exceeded the amount of full-length product despite extended growth periods up to eight generations. Therefore, when transcription is permitted through the TRS, Figure 7 (left panel), both parental strands must be rapidly repaired with a high probability before the next round of replication.
We observed that propagation of the plasmid with the insert of 50 repeats in the opposing orientation (Figure 7, right panel) was unaffected by transcription modulation of the lacZ gene. Interestingly, previous in vitro biophysical studies demonstrated that long tracts of CTG, CAG, GAC and GTC repeats formed hairpin structures (6–8,48,49) of variable stabilities. In addition, slippage rates declined in the order of GTC > GAC = CAG > CTG, whereas hairpin loop stabilities ascended in the opposing order (8). In agreement with these in vitro assays, we observed small-length polymorphisms indicative of DNA slippage in vivo for GTC?GAC repeats (Figure 1B) at a higher threshold length than for the opposing orientation. Thus, both in vitro and in vivo studies predict that GTC repeats adopt more stable hairpin structures than GAC repeats. Therefore, our model (Figure 7, right panel) assumes that hairpin formation is less likely when the non-transcribed strand consists of GAC repeats.
Surprisingly, for the longer TRS lengths (at 70 repeats) in both insert orientations, large deletion events predominated the mutation spectra even early in the first growth cycle under the influence of basal transcription. Additionally, this effect was completely altered by inhibiting transcription through the TRS that resulted in increasing amounts of small-length products and maintenance of the full-length tract as the predominant product. Thus, the longer repeat tracts, above the threshold length of 50, may have greater hairpin stability not only on the non-transcribed strand but also on the transcribed strand as the DNA duplex re-anneals after transcription.
Biological implications
These investigations on the genetic instability behaviors of repeating GAC?GTC tracts as influenced by transcription lay the initial groundwork for future work in other model systems. Since expansions and deletions of this previously unstudied TRS in the E.coli system could only be observed for repeats of 27 units and longer (up to 74 units), the development of yeast, cell culture or mouse models may be required to extend this work to the 5mer and related lengths to enable conclusions bearing more directly on the etiology of MED and PSACH. However, we anticipate that the general observations from our determinations (especially the transcriptional modulation of the repeats leading to partitioning of small- and large length products) will be relevant. Furthermore, it is possible that other hereditary neurological diseases (1,2) may be shown in the future to involve lengths of GAC?GTC repeats in the range utilized herein.
ACKNOWLEDGEMENTS
We thank Drs Albino Bacolla, Marek Nabievala and Adam Jaworski for helpful discussions and reading of the manuscript. This research was supported by National Institutes of Health grants NS37554 and ES11347 and from the Robert A. Welch Foundation.
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* To whom correspondence should be addressed. Tel: +1 713 677 7651; Fax: +1 713 677 7689; Email: rwells@ibt.tamu.edu
ABSTRACT
The genetic instability of (GAC?GTC)n (where n = 6–74) was investigated in an Escherichia coli-based plasmid system. Prior work implicated the instability of a (GAC?GTC)5 tract in the cartilage oligomeric matrix protein (COMP) gene to the 4, 6 or 7mers in the etiology of pseudoachondroplasia and multiple epiphyseal dysplasia. The effects of triplet repeat length and orientation were studied after multiple replication cycles in vivo. A transcribed plasmid containing (GAC?GTC)49 repeats led to large deletions (>3 repeats) after propagation in E.coli; however, if transcription was silenced by the LacIQ repressor, small expansions and deletions (<3 repeats) predominated the mutation spectra. In contrast, propagation of similar length but opposing orientation (GTC?GAC)53 containing plasmid led to small instabilities that were unaffected by the repression of transcription. Thus, by inhibiting transcription, the genetic instability of (GAC?GTC)49 repeats did not significantly differ from the opposing orientation, (GTC?GAC)53. We postulate that small instabilities of GAC?GTC repeats are achieved through replicative slippage, whereas large deletion events are found when GAC?GTC repeats are transcribed. Herein, we report the first genetic study on GAC?GTC repeat instability describing two types of mutational patterns that can be partitioned by transcription modulation. Along with prior biophysical data, these results lay the initial groundwork for understanding the genetic processes responsible for triplet repeat mutations in the COMP gene.
INTRODUCTION
The etiologies of approximately 14 inherited neurological disorders are due to the genetic instabilities of triplet repeat sequences (TRSs) . Three of the ten possible triplet repeats have been associated with the widely studied diseases such as myotonic dystrophy, Huntington's disease, spinocerebellar ataxias, fragile X syndrome and Friedreich's ataxia, which involve expansions of CTG?CAG, CGG?CCG and GAA?TTC repeats in the relevant genes (1,2). A fourth triplet repeat (GAC?GTC) was identified (3) in the cartilage oligomeric matrix protein (COMP) gene, which exhibited small expansions and deletions (1 or 2 repeats) from the parental 5mer. A single trinucleotide expansion leads to multiple epiphyseal dysplasia (MED), whereas two triplet expansions (or one repeat deletion) lead to pseudoachondroplasia (PSACH). MED and PSACH are clinically characterized by joint pain, short stature, early-onset osteoarthritis and limb dwarfism (3). Since a single TRS deletion or expansion is sufficient for disease onset, unstable GAC?GTC repeats contrast with other TRS-induced diseases that require larger or even massive TRS expansions to cause disease (1,2). Previously, GAC?GTC repeats were shown to be genetically unstable in a plasmid-based system (4), several years before this association was made to a disease state. In addition, comparative biophysical data (5–8) on synthetic oligonucleotides of GAC to CAG repeats as well as GTC to CTG repeats revealed similar hairpin folding properties in vitro. Thus, since the genetic instability of the CAG?CTG sequence has been intensely studied , we intended to evaluate whether these results would be applicable to GAC?GTC repeats.
Replication, recombination and DNA-repair-based mechanisms have been investigated to explain the genetic expansions of TRS in bacteria, yeast, cell culture and mammalian systems (1,9,10). Repeat instability is dependent on several factors, including the length and type of the TRS, the orientation of the repeats with respect to the direction of replication, the number of replication cycles and the genetic background of the host cells (9). These factors are not mutually exclusive. In addition, transcription was shown to cause increased levels of deletions in CAG?CTG repeats (11–13). One model for this behavior envisioned the non-transcribed strand adopting folded-back secondary structures in long CAG?CTG tracts; after several replication cycles, an increased frequency of deletions was observed in an orientation-dependent manner (12).
Herein, we investigated the mechanisms influencing instability in a plasmid-based system harboring various lengths of GAC?GTC repeats (6–74 repeats) to determine the types and amounts of expansions and deletions during multiple replication cycles in Escherichia coli. In addition, induction or repression of transcription through these repeats revealed that two types of mutational patterns, small and large length changes, are partitioned in an orientation- and length-dependent manner.
MATERIALS AND METHODS
Plasmids
The plasmids used in this study are listed in Table 1. Some plasmids containing GTC?GAC repeat tracts were constructed and characterized previously (14); pRW3413 (14) was used herein. Other GTC?GAC containing plasmids that were prepared and partially characterized (K. Ohshima and R. D. Wells, unpublished data) were sequenced; these included pRW3132, pRW3131, pRW3815 and pRW3820. Plasmids pRW4606, pRW4607 and pRW4608 are derivatives of pRW3413, pRW3815 and pRW3820, respectively. In order to clone the repeats in the opposing orientation, the inserts from pRW3413, pRW3820 and pRW3815 were released by HindIII and SacI digestion. Each insert from pRW3413, pRW3820 and pRW3815 was separated on a 7% acrylamide gel, stained with ethidium bromide and the TRS-containing bands were excised. The eluted DNAs from the excised bands were purified by phenol–chloroform extraction and precipitated with ethanol (15). The purified inserts were ligated to the corresponding linearized vector (pUC18 or pUC19) at approximately a 1:10 molar ratio with 20 U of T4 DNA ligase (United States Biochemical Corp.). The reactions were carried out at 16°C for 12 h. The ligated products were ethanol precipitated and transformed into E.coli HB101 by electroporation, and the transformed cells were plated on Luria–Bertani (LB) agar plates containing 50 μg/ml ampicillin (15). While cloning the inserts into the pUC vectors for the opposing orientation, most TRS-containing inserts were deleted by a few repeats.
Table 1. Plasmids used in this study
In order to attempt to obtain plasmids containing undeleted TRS inserts, each plasmid (Table 1) containing GAC?GTC and GTC?GAC repeats was electrophoresized on a 1% agarose gel. The corresponding band of supercoiled DNA was excised and eluted for phenol–chloroform extraction. After purification, the DNA was transformed into E.coli HB101, and the cells were spread onto an agar plate with ampicillin (50 μg/ml). Individual colonies were grown overnight at 37°C in liquid LB medium. DNAs from individual colonies were isolated by a Wizard Plus Minipreps DNA Purification System (Promega) and then analyzed by restriction mapping and sequencing. The length of the repeat from each plasmid (Table 1) was determined by digestion with HindIII and SacI to release the TRS-containing insert, and the fragments were radiolabeled with dATP (15) and resolved on a 7% native acrylamide gel. Dideoxy sequencing of both strands with a Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (United States Biochemical Corp.) was utilized for determining the sequence and orientation of the TRS-containing plasmid. The sequencing reactions were carried out according to the manufacturer's recommendations. The following primers were utilized: M13/pUC sequencing primer 5'd(GTAAAACGACGGCCAGT)3' (#S1211S New England Biolabs, Inc.) and M13/pUC reverse sequencing primer 5'd(AACAGCTATGACCATG)3' (#S1201S New England Biolabs, Inc.). The products of the sequencing reactions were analyzed on 6% acrylamide Long Ranger gels (FMC BioProducts) containing 8 M urea in glycerol tolerant buffer (United States Biochemical Corp.). The gels were dried and exposed to X-ray film.
Recultivation assay and product analyses
Each plasmid containing uninterrupted GAC?GTC and GTC?GAC repeat tracts were transformed into E.coli AB1157 . In order to obtain multiple recultivation experiments for each plasmid, three or more aliquots of each transformation mixture were grown overnight at 37°C in 10 ml liquid LB plus ampicillin (50 μg/ml) at a shaking rate of 250 r.p.m.; alternatively, a number of repeated transformations were performed for each plasmid. Each 10 ml liquid culture was designated as growth cycle 1. An aliquot of each growth cycle 1 culture was diluted 107-fold for inoculation into in a new 10 ml liquid LB plus ampicillin and grown overnight under the same conditions as the first inoculation. Recultivation of these cultures was repeated through four growth cycles. In addition, to determine the transformation efficiency, an additional aliquot from each transformation mixture was plated onto a 1.5% agar plate with ampicillin (50 μg/ml). Approximately 100–1000 transformants were obtained for each transformation mixture. After each growth cycle, the populations of cells were harvested and plasmid DNA was isolated. DNA from cultures (growth cycles 1–4) was digested with HindIII and SacI to release the repeat-containing inserts which were then radiolabeled with dATP for analyses by 7% native gel electrophoresis at 37°C.
For the experiments with inhibited transcription, the plasmid pIQ-kan was transformed into the E.coli AB1157 host strain. The pIQ-kan plasmid expresses the repressor for the lacZ promoter thereby inhibiting transcription of the lacZ gene. The TRS were cloned downstream of the lacZ promoter and therefore transcription through the TRS tract was also inhibited. In the presence of the repressor, the recultivation assay was carried out as described above with the addition of kanamycin (100 μg/ml) selection for the pIQ-kan plasmid. When required, isopropyl-?-D-thiogalactopyranoside (IPTG) of 1 mM was included in the growth medium for the induction of transcription. Replication and transcription through the repeat from the lacZ promoter for both pUC18 and pUC19 are co-directional (15).
After electrophoresis of the radiolabeled molecules from a recultivation experiment, the gel was exposed to a PhosphorImager screen (Molecular Dynamics). The gel was scanned and the areas under each peak were determined by using the Image Quant program (Molecular Dynamics).
RESULTS
The effect of repeat tract length on the instability of (GAC?GTC)n and (GTC?GAC)n in transcribed plasmids
The genetic stability of CAG?CTG, CGG?CCG and GAA?TTC repeat-containing constructs is dependent on the length of the repeat tracts (16–23). In order to determine the effect of length on the stability of (GAC?GTC)n and (GTC?GAC)n, the appropriate plasmids (Table 1) (where n ranged from 6 to 74) were propagated in a recultivation assay (18,24). The (GAC?GTC)n and (GTC?GAC)n sequences were cloned into the polylinker of pUC plasmids and are located 400 bp from the origin of replication for both pUC18 and pUC19. The (GAC?GTC)n and (GTC?GAC)n tracts, located in the lacZ gene which is under the control of the lacZ promoter, were transcribed. For our considerations herein, large deletion or large expansion events obtained during a recultivation assay were designated as >3 repeats and small deletion or small expansion events were designated as <3 repeats. After each growth cycle in a recultivation assay, plasmid DNAs were analyzed by HindIII–SacI digestion and the TRS-containing inserts were radiolabeled and resolved by native gel electrophoresis as described under Materials and Methods.
pRW3132 and pRW3131 were propagated in E.coli AB1157 for four growth cycles. Small deletion and expansion events were observed. However, the amounts of the instabilities were small (<10%) (data not shown). Thus, in order to enhance the instability in our genetic assay, we lengthened the TRS tract to evaluate the molecular mechanisms for GAC?GTC instability. pRW4606 and pRW3413 containing longer TRS tracts were propagated as described above. Figure 1A demonstrates that increasing growth cycles cause small expansions and deletions of the plasmids harboring (GAC?GTC)27 and (GTC?GAC)30 while the progenitor length of both molecules was maintained. After the recultivation study, DNA sequencing and restriction analyses of the products (Materials and Methods) revealed that both plasmids had deleted and expanded products with repeat lengths that were close to the progenitor repeat length. For example, the small deletion and expansion products consisted of lengths 27, 24, 22, 23, 17 and 15 repeats for pRW4606 and39, 36, 33, 31, 30, 28, 25, 24 and 23 repeats for pRW3413. These products were stable when propagated through subsequent growth cycles. Hence, these data show that expansions and deletions changed by only small increments in transcribed pRW4606 and pRW3413.
Figure 1. Analyses of in vivo genetic instabilities of (GAC?GTC)n and (GTC?GAC)n repeats. The results of a representative recultivation experiment through four growth cycles (indicated as 1–4) in E.coli AB1157 are shown; the plasmid DNA products were digested with HindIII and SacI, and the fragments, radiolabeled with dATP, were separated on a native 7% acrylamide gel as described under Materials and Methods. The arrowheads indicate the full-length fragments (no expansions or deletions) for each parental plasmid. The products of expansions and deletions are shown as a function of the growth cycles of plasmids containing (A) (GAC?GTC)27 and (GTC?GAC)30 repeats (B) (GAC?GTC)49 and (GTC?GAC)53 repeats and (C) (GAC?GTC)74 and (GTC?GAC)69 repeats.
pRW4608 and pRW3820, which are similar in length, with 20 more repeats than the DNAs described above, were also propagated in recultivation assays. The restriction analyses shown in Figure 1B demonstrate a loss of the full-length progenitor fragment with increasing growth cycles for pRW4608 and retention of the full-length fragment with increasing growth cycles for pRW3820. Furthermore, the pattern of genetic instabilities observed for the two molecules differ. For pRW4608, with a length of 49 repeats, the progenitor fragment is deleted entirely after the second growth cycle to give deletion products that are predominately large deletion events. The major instability events for pRW4608 had lengths of 61, 51, 49, 48, 46, 41, 32, 26, 19 and 14. Most of the length changes were >3 repeats. Alternatively, for pRW3820 with 53 repeats in the opposing orientation, the progenitor fragment was retained at its full length even after four growth cycles while generating small deletion and expansion events. The expansion and deletion products from pRW3820 consisted of lengths 63 and 59, all integral units from 57–47, 42, 40, 38, 34, 31 and all integral units from 30–19. Thus, the n was <3 for each member of this family of products. Hence, in actively transcribed plasmids, large deletion events predominated for pRW4608 and small expansion and deletion events predominated for pRW3820. From these data, the orientation of the insert was the major influencing factor in determining the type of instabilities obtained from pRW4608 and pRW3820.
Earlier studies (17,25–31) explained that a possible mechanism for the effect of insert orientation of CTG?CAG versus CAG?CTG repeats was due to the higher propensity of long CTG repeats to form a more stable hairpin structure than long CAG repeats on the lagging strand template. Thus, during replicative events, the single-stranded state of the lagging strand template allowed for preferential hairpin formation of CTG repeats than CAG repeats that could be consequently bypassed by DNA polymerase and subsequently lead to greater instability of CTG repeats than CAG repeats. Similar to CTG and CAG repeating sequences, we found that the mutational patterns were strongly dependent on insert orientation. For example, when the intensities of each band from Figure 1B was plotted as a function of migration distance (plot not shown), the peak corresponding to the progenitor full-length fragment containing (GAC?GTC)49 repeats diminished to baseline by growth cycle 3 and peak intensities corresponding to the large deletion products gained intensity with increasing growth cycles. However, in the opposing orientation, the intensity of the corresponding peak for pRW3820 was not only sustained throughout the four growth cycles, but the area of the base of the full-length peak broadened, indicating that small expansion and deletion events in the population were progressively accumulating near the full-length peak with successive growth cycles. Thus, pRW3820 containing (GTC?GAC)53 displayed the capacity to expand and delete by 1–3 repeats indicative of triplet repeat slippage during DNA replication, whereas the insert (GAC?GTC)49, more capable of hairpin formation, could render large deletion events due to possible hairpin bypass on the lagging strand template during DNA synthesis.
Plasmids with longer repeat tracts, (GAC?GTC)74 and (GTC?GAC)69, were also propagated for determining the extent of expansions and deletions. For both plasmids, the original full-length fragment from pRW3815 and pRW4607 were deleted entirely after the second growth cycle while generating large expansion and deletion products (Figure 1C). The products contained 77, 74, 71, 63, 55, 44, 38, 36, 35, 34, 22, 20, 18 and 15 repeats for pRW3815 and 69, 68, 64, 62, 44, 40, 34, 28, 23, 22, 20 and 16 repeats for pR4607.
In summary, these data show that GAC?GTC and GTC?GAC repeats are unstable in transcribed plasmids. Smaller length repeats (30 repeats) deleted and expanded by small increments whereas longer repeat lengths (70 repeats) deleted as large deletions. A length threshold was observed in the types of instabilities obtained after four growth cycles for pRW4608 and pRW3820 with 50 repeats. The mechanisms of the large instabilities from pRW4608 and the small instabilities from pRW3820 are further explored below by inhibiting transcription of the lacZ gene.
Quantitation of instability products from (GAC?GTC)n and (GTC?GAC)n
Figure 2 shows the percentages of expansions, deletions and full-length retentions of GAC?GTC and GTC?GAC tracts averaged from three independent recultivation experiments. Inserts in the GAC?GTC orientation with lengths of 27, 49 and 74 demonstrate that the percentage of the full-length repeat decreases as the length of the repeat sequence increases. In this orientation, expansion events were infrequent, but the percentage of deletions dramatically increased as the tracts lengthened from 27 to 74 repeats. By the third growth cycle for length 49, deletions were essentially the only type of mutation observed.
Figure 2. The amounts of products formed from (GAC?GTC)n and (GTC?GAC)n repeats as a function of growth cycles. The extents of genetic instabilities were determined by quantitating the areas of all peaks in each lane as described in Materials and Methods. The averages from three independent recultivation experiments (including experiments from Figure 1) of percentage expansions (filled bars), deletions (gray bars) and original length fragments (crosshatched bars) are plotted against the number of growth cycles (indicated as 1–4). The length and orientation of the inserts are denoted above each plot.
In the orientation GTC?GAC (Figure 2, right-hand side), the percentage of full-length progenitor fragment was sustained for pRW3413 containing length 30 and for pRW3820 containing length 53, but not for pRW3815 containing length 69. For length 30, the full-length fragment only diminishes by 18% from the initial growth cycle to the end of the fourth growth cycle whereas in the other orientation for pRW4606 containing length 27, the percentage is reduced by 74%. In addition, expansion events for pRW3413 with 30 repeats constituted 7% of the genetic instability that was sustained throughout the four growth cycles. With the same insert orientation but with the longer repeat, pRW3820 containing (GTC?GAC)53, the full-length fragment was reduced by 44% after four growth cycles and the expansion events constituted 10% of the overall percentage. As the length of the repeat array increases, for plasmids with similar length inserts , there is a loss of the full-length fragment by the third growth cycle from 37 to 0% and from 41 to 0%, respectively; thus, the effect of orientation was overcome by length.
Hence, these data show that the instabilities of the GAC?GTC and GTC?GAC repeats are dependent on the length of the tract. The rapid loss through the growth cycles of the full-length fragment in the GAC?GTC orientation compared to the GTC?GAC orientation indicates that GAC?GTC repeats deleted more readily than GTC?GTC repeats. In addition, the TRS mutations in the GTC?GAC orientation exhibited a significant percentage of expansions in addition to deletions.
Repression of transcription influences the types of genetic instabilities in (GAC?GTC)49 and (GTC?GAC)53 tracts
Prior investigations have shown that transcription through repetitive DNA increases the occurrence of deletions in bacteria (11–13,32,33) and in yeast (34). Two types of genetic instability patterns described as large deletion and expansion and small deletion and expansion events had been described previously in plasmids containing CTG?CAG repeats (24,35–37). Similarly, two different mutational patterns appeared in the recultivation of plasmids containing (GAC?GTC)49 and (GTC?GAC)53 repeats when transcription and replication were coupled (see above).
In order to determine the genetic instabilities arising from DNA replication-mediated events in GAC?GTC and GTC?GAC repeats, transcription was silenced. Transforming the pIQ-kan plasmid that synthesizes the LacIQ repressor into the E.coli AB1157 host inhibited transcription. The LacIQ repressor inhibits transcription from the lacZ promoter. Conversely, the addition of IPTG, a gratuitous inducer, stimulates transcription (32). After electroporation of the pIQ-kan plasmid into E.coli AB1157, the cells were additionally transformed with either (GAC?GTC)49 or (GTC?GAC)53 containing plasmids. The cultures were allowed to pass through four growth cycles, and the cells were harvested at approximately every 20 generations (one growth cycle) as described above.
Figure 3A shows the results of restriction analyses of pRW4608 and pRW3820 during four growth cycles. Large deletion events were dramatically reduced compared to data in the presence of basal transcription (Figure 1B) during the recultivation of pRW4608 in the presence of LacIQ repressor; the products consisted of small expansions and deletions as the predominant types of mutagenic events. pRW4608 was subcultured for eight generations to test whether the starting fragment length was also deleted; Figure 3 shows that the progenitor length was sustained as the predominant product (data from growth cycles 5–8 not shown). In contrast, the recultivation of pRW3820 under the same conditions did not show a significant alteration in the small deletion and expansion pattern as observed previously in the presence of transcription (Figure 1B), thus indicating that the silencing of transcription did not influence the instability of pRW3820. The percentages of the progenitor full-length fragment are diminished by only 8% for pRW4608, whereas pRW3820 was deleted by 31% by the end of four growth cycles (Figure 4A). Thus, large deletions in (GAC?GTC)49 are associated with transcription.
Figure 3. The effects of transcription silencing on the genetic instabilities of (GAC?GTC)49 and (GTC?GAC)53 repeats. E.coli AB1157 harboring pIQ-kan was transformed with the supercoiled monomers of the designated plasmids. The instability of the (GAC?GTC)49 and (GTC?GAC)53 inserts were analyzed as described in the legend to Figure 1 and in Materials and Methods. The growth cycles are indicated as 1–4. The full-length progenitor fragments of the each parental plasmid are indicated by an arrowhead. (A) The results from a representative recultivation experiment of pRW4608 and pRW3820 that were carried out in the presence of LacIQ repressor for determining the extent of genetic instabilities derived from replication-mediated events while lacZ gene transcription was inhibited. (B) The results from a recultivation experiment with pRW4608 and pRW3820 as a function of four growth cycles in the presence LacIQ repressor and IPTG.
Figure 4. The effects of transcription silencing on the percentage of genetic instabilities of (GAC?GTC)49 and (GTC?GAC)53 repeats. Instability studies similar to the data in Figure 3 were quantitated as described in Materials and Methods. (A) The extent of instabilities of pRW4608 and pRW3820 in E.coli AB1157 in the presence of the LacIQ repressor is expressed by percentages of expansions (filled bars), deletions (gray bars) and original full-length material (crosshatched bars) and plotted against multiple growth cycles (indicated as 1–4). (B) The percentages of genetic instabilities of pRW4608 and pRW3820 when transcription is restored by the addition of IPTG and LacIQ repressor. The percentages are averages of three individual experiments including Figure 3.
To confirm whether large deletions are caused by transcription-mediated events in the TRS of pRW4608, IPTG was used to overcome repression by the LacIQ repressor and thus induce transcription. As stated above, recultivations of pRW4608 and pRW3820 were carried out in parallel with the addition of IPTG in the growth medium for four growth cycles. As expected (Figure 3B), large deletion events were found during the four growth cycles of pRW4608 causing 5-fold destabilization of the progenitor full-length fragment (compared with data in Figure 3A). However, in addition to the large deletion events observed for pRW4608, the progenitor full-length fragment was also sustained throughout the four growth cycles with the addition of IPTG. The presence of both types of instabilities was probably due to either greater amounts of repressor molecules than IPTG molecules in certain cells, or, alternatively, the repressor molecules may have greater binding affinity to the lacZ promoter than to IPTG under these growth conditions. In support of the latter case, in a separate control experiment (data not shown), we observed that large deletions were not inducible when IPTG was introduced late (in growth cycles 5–8) in the presence of repressor during the recultivation of pRW4608. Thus, we conclude that LacIQ repressor is an effective inhibitor of large deletions despite the high amounts of IPTG molecules. In the opposing orientation, the IPTG induction of transcription did not affect the small expansion and deletion events which were observed previously with pRW3820 in Figure 3A (right-hand side).
Repression of transcription influences the genetic instabilities of TRS longer tracts
We hypothesize that large deletion events arise through a transcription-mediated mechanism involving DNA loop structures, whereas small expansion and deletion events are the result of a TRS slippage during DNA replication. To evaluate our hypothesis, the propagation of plasmids containing the longer lengths of (GAC?GTC)74 and (GTC?GAC)69 repeats should result in small deletion and expansion mutations if transcription were silenced rather than giving large deletion events as previously shown in Figure 1C. To test this hypothesis, pRW3815 and pRW4607, which harbor longer repeat lengths, were also grown for four growth cycles in the presence of the LacIQ repressor (Figure 5A). With transcription blockage (Figure 5B), both molecules sustained 30 and 34%, respectively, of the progenitor length fragment by the end of four growth cycles. In addition, small instabilities were observed where the full-length fragment was altered to lengths of all integral units from 73–67, 63, 60, 56, 53, 51, 47, 44, 42, 41, 39, 36, 33, 29, 26, 24, 21 from plasmids containing (GAC?GTC)74 repeats and to lengths of all integral units from 68–48, 46–43, 40, 38, 34, 30, 29, and 25, 24, 23 from plasmids containing (GTC?GAC)69 repeats. The majority of the length changes were <3 repeat units indicative of slippage during DNA replication. Hence, these results show that the inhibition of transcription not only stabilizes the parental plasmid even in longer TRS arrays but that transcription through the repeats affects the types of genetic instabilities that include large deletions when transcription is turned on to small deletions and expansions when transcription is turned off.
Figure 5. The effects of transcription silencing on the longer repeat tracts of (GAC?GTC)74 and (GTC?GAC)69. E.coli AB1157 harboring pIQ-kan was transformed with pRW3815 and pRW4607 for determining the influence of transcription through longer TRS repeats. (A) Through four recultivations, the DNAs were isolated and the TRS-containing inserts were released with HindIII and SacI digestion. The figure shows a 7% native gel electropherogram of the products through four growth cycles (indicated as 1–4) when transcription was inhibited by the LacIQ repressor. Arrowheads mark the full-length fragments derived from the parental plasmids. (B) The percentage of genetic instability is displayed for pRW3815 and pRW4607 through the four growth cycles. The expansions (filled bars), deletions (gray bars) and original progenitor full-length fragments (crosshatched bars) are plotted against the number of growth cycles. The percentages are averages of three individual experiments including the data in (A).
DISCUSSION
Two types of expansion and deletion products were partitioned by modulating transcription through GAC?GTC repeats in an E.coli model system. Small expansions and deletions in descending amounts relative to the full-length tract were observed for TRS lengths of <30 repeats and for (GTC?GAC)53 repeats under basal transcription conditions. In the opposing orientation, (GAC?GTC)49, the pattern of instabilities was different; large deletion products predominated in the population while the progenitor full-length tract was entirely deleted. However, if transcription was inhibited, the products were small expansions and deletions with the maintenance of the progenitor full-length tract. Interestingly, induction or inhibition of transcription through the (GTC?GAC)53 repeats did not alter the pattern or extent of small expansions and deletions indicating a threshold length in the type of instabilities obtained after successive growth cycles. Furthermore, the longer TRS lengths (>69 repeats) also generated large deletion products in both insert orientations but products could also be altered by inhibition of transcription. Thus, transcription through the repeats promotes large deletion products in long repeat tracts in an orientation-dependent manner; in contrast, inhibition of transcription through the repeats led to enhancement of small expansions and deletions independent of insert orientation.
Replication-based instability of (GAC?GTC)n and (GTC?GAC)n
Figure 6 shows a model to explain the occurrence of small instabilities; the length changes are proposed to occur during the discontinuous synthesis of the Okazaki fragments from the lagging strand template rather than during the continuous synthesis of the leading strand. During Okazaki fragment processing, the single-strand state of the DNA facilitates the formation of slipped structures (38,39). We propose that during DNA replication, the original length, N, would be conserved on the leading strand template , while misalignment of the repeat on the lagging strand template , would cause length changes. In the latter case, DNA polymerase would bypass the looped-out repeats from the lagging strand template that could potentially be carried through to the next round of replication . Thus, the new leading strand would have a deletion of one repeat from the original length and the new lagging strand would retain the progenitor length (N).
Figure 6. Model for DNA slippage during replication of GAC?GTC and GTC?GAC repeats when transcription is silenced. During DNA replication, the lagging strand synthesis allows for single-stranded regions to misalign, either on the lagging strand template (A) or on the lagging nascent strand (B) in the regions of the repeating tract. (A) While in (a), the leading strand template (top strand of fork) retains the progenitor length (N), in (b) DNA slippage occurs by one repeat on the lagging strand. The DNA polymerase complex is proposed to bypass the looped-out repeat on the lagging strand template (17). (c) In the subsequent replication cycle, the new leading strand template has a deletion of 1 repeat (N – 1) whereas the new lagging strand template retains the progenitor repeat length (N). (B) (a) is the same as described for (A). (d) If the slippage of 1 repeat occurs on the nascent lagging strand, (e) then the newly synthesized top strand in the subsequent replication cycle will have an expansion of one repeat (N + 1) while the lower lagging strand template retains the progenitor length (N) (17,24).
Alternatively, if synthesis in excess of one repeat unit (expansions) occurs by misalignment on the lagging nascent strand , then in the subsequent replication event the new leading strand would consist of an expansion (N + 1) and the new lagging strand would conserve the progenitor full-length fragment (N). Our data show that the progenitor full-length fragment was sustained throughout multiple growth cycles as the predominant product while accumulating small expansion and deletion products of descending lengths and intensities, particularly when the repeats were not transcribed (Figure 3A). Thus, this model would explain the probability of maintaining the full-length fragment in higher amounts than the expanded and deleted products.
Transcription-associated mutations in the repeats
Transcription into tracts of the myotonic dystrophy CTG?CAG repeats enhanced the instability of the repeats, especially deletions (11,13). We previously proposed a model that envisioned transcription-induced local positive and negative superhelical domains that could facilitate hairpin formation of the CTG?CAG repeats (12) on the non-transcribed strand. Figure 7 extends this model with the consideration of the effect of insert orientation when transcription occurs through similar length repeats (left-hand side), resulting in two types of mutational patterns. After the DNA duplex melting by RNA polymerase (40) and resulting negative supercoil unwinding, the DNA is unwound (41,42). The transient single-stranded state of the non-transcribed strand containing GTC repeats enables quasi-stable hairpin formation. After passage of the RNA polymerase complex and during DNA re-annealing, any hairpins on the non-transcribed strand may generate several unpaired bases on the complementary strand. Therefore, these unpaired bases may transiently remain in an unstable looped-out structure. These loci may be substrates for mismatch repair or NER . Prior studies with the E.coli LacI (44,45) and human hprt genes (46) showed that the unpaired non-transcribed DNA strand had a greater propensity for mutations than the transcribed strand. Furthermore, the non-transcribed strand was repaired 4 times slower than the transcribed strand in the human p53 gene (47). Thus, the non-transcribed strand may have a greater propensity to be mutated than the template strand due to the transient single-stranded state.
Figure 7. Model for the involvement of transcription in the instabilities of GAC?GTC and GTC?GAC repeats of an intermediate length. RNA polymerase causes local duplex melting at the TRS tract for the initiation of transcription (40). Local positive and negative superhelical domains aid the progression of the RNA polymerase through the duplex DNA (41,50). While the transcribed GAC strand (left panel) complexes with the newly formed mRNA, the GTC strand which is not transcribed can readily form a hairpin. Re-annealing of the duplex DNA after transcription termination allows for the remaining unpaired bases on the GAC strand to loop out. The hairpin or looped-out structures are potential substrates for DNA repair proteins such as those that are involved in MMR (43), NER (51,52) or the SOS response (43). It is proposed that the structures are excised, DNA polymerase fills-in the gaps and the nicks are sealed by ligase. The removal of several bases due to their engagement as a hairpin or looped-out structure leads to large deletions on both strands of the DNA. In the other TRS orientation (right panel), when the GTC strand pairs with the mRNA, the GAC strand becomes unpaired; however, the GAC strand at an intermediate length does not form a hairpin structure as readily as the GTC unpaired strand (left panel). Therefore, transcription through intermediate-length GTC?GAC repeats does not stimulate genetic instabilities.
In previous studies (11,12), when CTG?CAG repeats were propagated in E.coli HB101, the main determinant for the loss of progenitor full-length tract was the increased growth rates of bacterial cells harboring deleted products. Similarly, we found that transcription through (GAC?GTC)49 led to the complete loss of the progenitor full-length tract with increasing amounts of large deletions. However, this interpretation was not plausible when transcription was inhibited through the repeat. Instead, we observed that the progenitor full-length tract was present in higher amounts than the expanded and deleted products. Furthermore, the small length changes never exceeded the amount of full-length product despite extended growth periods up to eight generations. Therefore, when transcription is permitted through the TRS, Figure 7 (left panel), both parental strands must be rapidly repaired with a high probability before the next round of replication.
We observed that propagation of the plasmid with the insert of 50 repeats in the opposing orientation (Figure 7, right panel) was unaffected by transcription modulation of the lacZ gene. Interestingly, previous in vitro biophysical studies demonstrated that long tracts of CTG, CAG, GAC and GTC repeats formed hairpin structures (6–8,48,49) of variable stabilities. In addition, slippage rates declined in the order of GTC > GAC = CAG > CTG, whereas hairpin loop stabilities ascended in the opposing order (8). In agreement with these in vitro assays, we observed small-length polymorphisms indicative of DNA slippage in vivo for GTC?GAC repeats (Figure 1B) at a higher threshold length than for the opposing orientation. Thus, both in vitro and in vivo studies predict that GTC repeats adopt more stable hairpin structures than GAC repeats. Therefore, our model (Figure 7, right panel) assumes that hairpin formation is less likely when the non-transcribed strand consists of GAC repeats.
Surprisingly, for the longer TRS lengths (at 70 repeats) in both insert orientations, large deletion events predominated the mutation spectra even early in the first growth cycle under the influence of basal transcription. Additionally, this effect was completely altered by inhibiting transcription through the TRS that resulted in increasing amounts of small-length products and maintenance of the full-length tract as the predominant product. Thus, the longer repeat tracts, above the threshold length of 50, may have greater hairpin stability not only on the non-transcribed strand but also on the transcribed strand as the DNA duplex re-anneals after transcription.
Biological implications
These investigations on the genetic instability behaviors of repeating GAC?GTC tracts as influenced by transcription lay the initial groundwork for future work in other model systems. Since expansions and deletions of this previously unstudied TRS in the E.coli system could only be observed for repeats of 27 units and longer (up to 74 units), the development of yeast, cell culture or mouse models may be required to extend this work to the 5mer and related lengths to enable conclusions bearing more directly on the etiology of MED and PSACH. However, we anticipate that the general observations from our determinations (especially the transcriptional modulation of the repeats leading to partitioning of small- and large length products) will be relevant. Furthermore, it is possible that other hereditary neurological diseases (1,2) may be shown in the future to involve lengths of GAC?GTC repeats in the range utilized herein.
ACKNOWLEDGEMENTS
We thank Drs Albino Bacolla, Marek Nabievala and Adam Jaworski for helpful discussions and reading of the manuscript. This research was supported by National Institutes of Health grants NS37554 and ES11347 and from the Robert A. Welch Foundation.
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