The nature of actinomycin D binding to d(AACCAXYG) sequence motifs
http://www.100md.com
《核酸研究医学期刊》
Department of Chemistry, Tennessee State University, Nashville, TN 37209–1561, USA, 1 Institute of Biochemistry, National Chung-Hsing University, Taichung 40227, Taiwan and 2 Department of Life Science, National Central University, Jung-Li 320, Taiwan
*To whom correspondence should be addressed. Tel: +1 615 963 5325; Fax: +1 615 963 5434; Email: fchen@tnstate.edu
ABSTRACT
Earlier studies by others had indicated that actinomycin D (ACTD) binds well to d(AACCATAG) and the end sequence TAG-3' is essential for its strong binding. In an effort to verify these assertions and to uncover other possible strong ACTD binding sequences as well as to elucidate the nature of their binding, systematic studies have been carried out with oligomers of d(AACCAXYG) sequence motifs, where X and Y can be any DNA base. The results indicate that in addition to TAG-3', oligomers ending with XAG-3' and XCG-3' all provide binding constants 1 x 107 M–1 and even sequences ending with XTG-3' and XGG-3' exhibit binding affinities in the range 1–8 x 106 M–1. The nature of the strong ACTD affinity of the sequences d(A1A2C3C4A5X6Y7G8) was delineated via comparative binding studies of d(AACCAAAG), d(AGCCAAAG) and their base substituted derivatives. Two binding modes are proposed to coexist, with the major component consisting of the 3'-terminus G base folding back to base pair with C4 and the ACTD inserting at A2C3C4 by looping out the C3 while both faces of the chromophore are stacked by A and G bases, respectively. The minor mode is for the G to base pair with C3 and to have the same A/chromophore/G stacking but without a looped out base. These assertions are supported by induced circular dichroic and fluorescence spectral measurements.
INTRODUCTION
Actinomycin D (ACTD) is an anticancer antibiotic best known for inhibiting transcription by binding to double-stranded DNA (1–5). It is a chromopeptide consisting of a phenoxazinone planar chromophore with two pentapeptide rings attached. Its mode of DNA binding has been shown to be intercalative, in which the chromophore inserts in between the DNA base pairs while the two chains of the pentapeptide rest in the minor groove. The GpC sequence specificity of this drug has been well characterized and originates from its ability to form two hydrogen bonds between each guanine and a pentapeptide ring (6,7).
However, there have been reports to indicate that this drug can also bind strongly to some non-GpC-containing sequences (8–10) as well as to some single-stranded (ss)DNA (11–16). Studies in our laboratory have discovered that ACTD binds strongly to the sequence d(TGTCATTG) of apparent single-stranded conformation and without GpC sites (17). A fold-back binding model was speculated wherein the planar phenoxazone inserts at the GTC site with a looped out T base whereas the 3'-terminus G folds back to form a base pair with the internal C and stacks on the opposite face of the chromophore. Additional supports for the hairpin binding model came from systematic binding studies with oligomers of sequence motifs d(TGTCTnG) and d(TGTnGTCT) that are devoid of GpC sequences and exist predominantly in single-stranded conformation (18).
Our recent NMR studies on the complex structure of ACTD bound to hairpins containing the DNA recognition site 5'-GXC/CYG-5' in the duplex stem (where X/Y is a G·C or T·A Watson–Crick base pair) further revealed a rather unusual interaction mode for the intercalated drug, in which the central Watson–Crick X/Y base pairs are disrupted and displaced by the ACTD chromophore (19). The looped out bases are not disordered but interact perpendicularly with the base/chromophore and form specific hydrogen bonds with DNA. These results support the binding principle that the ACTD chromophore prefers to be stacked on each side by the 3'-end of the G and demonstrate the important roles played by the looped out bases. Based on such a principle, a strong ACTD binding sequence of seemingly ssDNA oligomer 5'-CCGTT3GTGG-3' was designed and its high quality NMR spectra allowed us to determine its ACTD complex structure (20). The DNA oligomer 5'-CCGTT3GTGG-3' forms a hairpin structure in the complex, with tandem G·T mismatches in the stem region next to a loop of three stacked thymine bases pointing towards the major groove. Bipartite hydrogen bonds were detected for the G·T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ACTD intercalates nicely between the tandem G·T mismatches in essentially one major orientation. The hydrophobic G/phenoxazone/G interaction in the ACTD/5'-CCGTT3GTGG-3' complex was found to be more robust than that of the classical ACTD/5'-GpC-3' complex, consistent with the roughly 2-fold stronger binding of ACTD with 5'-CCGTT3GTGG-3' than with its 5'-CCGCT3GCGG-3' counterpart.
Although other hairpin models for single-stranded ACTD binding had earlier been suggested, the sequence context appeared to be quite distinct (15,16,21). Systematic binding studies of 7-amino-actinomycin D (7-AM-ACTD) with single-stranded oligomers containing -TAGT- sequences led to the suggestion that the drug binds to metastable hairpins. The hairpins bound most tightly by ACTD appear to contain non-Watson–Crick base pairs, including an A/G and two T/T mismatches (15). Studies by Rill’s group, however, suggested that binding of ACTD to ssDNA might not necessarily require DNA secondary structure at the outset (16,21). Oligonucleotide d(GTTAACCATAG) was identified as one of the rare ssDNAs that binds ACTD with high affinity. Although this sequence does not appear a priori favorable for haripin formation, NMR and other studies have suggested that in the ACTD complex the oligonucleotide adopts a stable, folded hairpin with an ACCA loop and 5'-TTA/GAT-5' stem (21). A more systematic binding study (16) was then carried out on a series of oligonucleotides corres ponding to progressive shortening of d(GTTAACCATAG) from the 5'-end plus a large number of related sequences. A minimum sequence of d(AACCATAG) was found sufficient for tight binding and indeed exhibits the highest ACTD affinity among the oligomers studied. It was also concluded that a TAG-3' terminal sequence is essential for strong ACTD binding. These results support the hypothesis that the initial binding of ACTD to certain ssDNA sequences can induce a substantial conformational change in the flanking sequences to achieve a highly stable ssDNA/ACTD complex and show that mechanisms of ACTD binding to DNA are more diverse than commonly perceived.
Elucidation on the nature of ACTD binding to d(AACCATAG) is important and could provide additional insight into the action of this drug, since it is devoid of G/chromophore/G stacking in complex formation. To this end, our laboratory has now carried out systematic ACTD binding studies with d(AACCAXYG) and related sequences. The results are presented herein and a binding model is proposed to account for the observed binding behaviors.
MATERIALS AND METHODS
Synthetic oligonucleotides were purchased from ResGen (Huntsville, AL) and used without further purification. These oligomers were purified by the vendor using reverse phase oligonucleotide purification cartridges and exhibited single band electrophoretic mobilities in denaturing polyacrylamide gel electrophoresis with stated purity of 95%. Concentrations of the DNA solutions (in nucleotides) were determined by measuring the absorbances at 260 nm after melting (at 95°C). The extinction coefficients of DNA oligomers were obtained via nearest neighbor approximation using mono- and dinucleotide values tabulated in Fasman (22). ACTD and 7-AM-ACTD were purchased from Serva. Concentrations of the drug solutions were determined by measuring the absorbances at 440 nm (for ACTD) and 528 nm (for 7-AM-ACTD), using extinction coefficients of 24 500 and 23 600 cm–1 M–1, respectively. Stock solutions of oligonucleotides and drugs were prepared by dissolution in 10 mM Tris–borate buffer pH 8 containing 0.1 M NaCl and 1 mM MgCl2. Absorption spectra were measured with a Cary 1E spectrophotometric system. Absorption spectral titrations were carried out starting with 2 ml of a 5 μM ACTD solution followed by progressive additions of the oligomer stock at equal time intervals. Absorbance differences between 427 and 480 nm during absorption spectral titrations were used to obtain the binding isotherms. Fluorescence spectra were obtained with a SLM 48000S system at room temperature using a cell of 1 cm path length. Circular dichroic (CD) spectra were measured at room temperature with a Jasco J-500A recording spectropolarimeter using water-jacketed cylindrical cells of 2 cm path length.
Association binding constants (K) were extracted via non-linear least squares fits on the experimental binding isotherms using a 1:1 drug to strand binding model: D + S = DS, where D, S and DS are free drug, free single-stranded DNA and drug–DNA complex, respectively. By means of equations for the mass balances of drug and DNA (in strand), the following equations can be derived:
D2K + D = Dt
S = Dt(X – 1) + D
Y = (D + 1 K S)D/Dt
where X St/Dt, D and 1 are the extinction coefficients of the free and bound drugs and Dt and St are the total drug and DNA strand concentrations, respectively, at each point of the titration. Experimental binding isotherms were plotted as the apparent extinction coefficient (Y) versus X and non-linear least squares fits to the above equations were made by initially fixing the known D and subsequently relaxing it to obtain the best fit to extract the binding constant K. The non-linear least squares fit program in Micromath (Salt Lake City, UT) was used for our fitting purpose.
RESULTS
Is the TAG sequence at the 3'-end of d(AACCATAG) essential for its strong ACTD binding?
In an effort to ascertain the validity of the assertion of a TAG-3' requirement in the strong ACTD binding of d(AACCATAG), systematic ACTD binding studies were made with oligonucleotides with d(AACCAXYG) sequence motifs, where X and Y can be any base. The results are shown in Table 1. These binding constants were extracted via non-linear least squares fits using a 1:1 drug to strand binding model on the experimental data points. Some representative binding isotherms and the goodness of fit can be seen in Figure 1, with their significance to be discussed later. It is found that in addition to TAG, sequences such as AAG, GAG, and CAG, as well as ACG, TCG, GCG and CCG, at the 3'-end all provide very strong ACTD binding, exhibiting binding constants 1 x 107 M–1. However, significant ACTD binding affinities are also exhibited by the rest of the sequences, with binding constants ranging from 1 to 8 x 106 M–1. These results suggest that the trinucleotide sequences XYG at the 3'-end can provide strong ACTD binding if preceded by AACCA at the 5'-end. It is noteworthy that oligomers with XAG and XCG endings exhibit decidedly stronger ACTD affinities than the corresponding XTG- or XGG-containing sequences.
Table 1. Comparison of ACTD binding constants at 25°C for the d(AACCAXYG) oligomers
Figure 1. Equilibrium binding isotherms for ACTD binding to d(AACCAAAG) and selective oligomers derived by single base substitution. Absorbance spectral titrations were carried out at 25°C and the absorbance difference A is that between 427 and 480 nm. Concentrations of both ACTD and DNA (in strands) are expressed in μM. Solid curves are those of non-linear least squares fits of the experimental isotherms using the 1:1 drug to strand binding model.
The G base at the 3'-end is vital for strong ACTD binding
To elucidate the nature of and to uncover the crucial DNA bases responsible for the strong ACTD binding of these oligomers, further studies were made with related oligonucleotides via base substitutions in d(AACCAAAG), a sequence which is seen to bind as strongly to ACTD as d(AACCATAG) and exhibits a binding constant of 10 x 106 M–1 (see Table 1). The most obvious starting point is to find out if a G base in the sequence is essential for binding, as it has the capacity to form hydrogen bonds with the drug threonine bases in the pentapeptide rings. Thus, oligomers with the 3'-terminal G replaced by A, T or C were studied and the results indicate that d(AACCAAAA), d(AACCAAAT) and d(AACCAAAC) all exhibited binding constants <<1 x 105 M–1, at least two orders of magnitude weaker than the parent oligomer containing the G base (see Table 2). These results indicate that the G base at the 3'-end is vital for the strong ACTD binding observed with these oligomers.
Table 2. Comparison of ACTD binding constants at 25°C for some oligomers derived from base substitutions of d(AACCAAAG)
Both C bases in AACC are essential but C4 is significantly more important than C3
To assess the contributions made by the two C bases as well as to compare their relative importance, ACTD binding studies were carried out with base replacement of either of the C residues in d(A1A2C3C4A5A6A7G8) and the results are also included in Table 2. Binding constants of 0.64, 1.2 and 0.71 x 106 M–1 were found for d(AAACAAAG), d(AATCAAAG) and d(AAGCAAAG), respectively, whereas values of 0.11, 0.15 and 0.18 x 106 M–1 were found for d(AACAAAAG), d(AACTAAAG) and d(AACGAAAG), respectively. Although considerable reductions in binding affinity are observed for base substitutions at either position, replacements of the C4 base are seen to be decidedly more affective. This suggests that both C bases contribute substantially, but that C4 appears to make a considerably (several-fold) more important contribution to the strong ACTD binding of these oligomers.
Replacement of the A base at the 5'-terminus does not greatly affect binding but its removal does
The roles played by the two A bases at the 5'-end were assessed by binding studies with base substitution of either of them and the results are also shown in Table 2. ACTD binding constants for d(TACCAAAG), d(GACCAAAG) d(CACCAAAG) were found to be 7.8, 15 and 4.8 x 106 M–1, respectively. Thus, base replacement of the terminal A does not greatly affect the binding strength. On the other hand, removal of the 5'-terminal base resulting in the heptamer d(ACCAAAG) diminishes the binding affinity by 20-fold.
Replacement of A2 by pyrimidines greatly diminishes while replacement with G base significantly enhances ACTD binding
A very different trend is observed when A2 is replaced by another DNA base. ACTD binding constants for d(ATCCAAAG), d(AGCCAAAG) and d(ACCCAAAG) were found to be 0.3, 100 and 0.4 x 106 M–1, respectively. Thus, substitution with a pyrimidine base T or C resulted in more than an order of magnitude reduction in the binding affinity. In contrast, a G base substitution resulted in a considerably higher binding strength. Such an enhancement upon G base substitution, coupled with the diminution of binding affinity upon pyrimidine substitution, provide us with considerable insights as to the possible modes of ACTD binding to the d(AACCAXYG) oligomers.
Binding studies with oligomers derived from base substitution in d(AGCCAAAG)
The enhanced ACTD binding of d(AGCCAAAG) provides us with a good road map for delineating the nature of ACTD binding to d(AACCAAAG), as the binding mode of the former can more easily be deduced due to the presence of an extra G at a strategic position in the sequence for possible utilization of the G/chromophore/G binding principle. To this end, ACTD binding studies were made with oligomers derived by base replacement in d(AGCCAAAG) and some of the results are shown in Table 3 (results on base replacement in A5A6A7 are not included as no dramatic variation was observed). The critical role played by the 3'-terminus G base is again illustrated by the much-weakened ACTD binding affinity when this base is replaced. Despite the presence of GpC sites, ACTD binding constants for d(AGCCAAAA), d(AGCCAAAT) and d(AGCCAAAC) were found to be 0.05, 0.08 and 0.07 x 106 M–1, respectively. These values are more than three orders of magnitude weaker than the parent octamer d(AGCCAAAG). In contrast, base replacement of the 5'-terminus A base appears to have minimal effect on the affinity for ACTD, as demonstrated by the high binding constants of 70, 100 and 36 x 106 M–1 retained by d(TGCCAAAG), d(GGCCAAAG) and d(CGCCAAAG), respectively. Base replacement of C3 (so that only C4 can form a base pair with G) resulted in d(AGACAAAG), d(AGTCAAAG) and d(AGGCAAAG), which exhibit ACTD binding affinities of 15, 8 and 10 x 106 M–1, respectively. This is still very strong binding, despite the fact that they are about an order of magnitude weaker than the 100 x 106 M–1 exhibited by the parent oligomer d(AGCCAAAG). In fact they are several-fold stronger than those obtained via base replacement at C4 (so that only C3 can now form a base pair with G) to result in d(AGCAAAAG), d(AGCTAAAG) and d(AGCGAAAG), which exhibit binding constants of 0.6, 1.8 and 2.4 x 106 M–1, respectively. These results suggest that C4 plays a more pivotal role than C3 during ACTD binding, a result akin to what was found for the d(AACCAAAG) counterparts.
Table 3. Comparison of ACTD binding affinities at 25°C for some oligomers derived from base substitutions of d(AGCCAAAG).
Representative binding isotherms
Selective ACTD binding isotherms illustrating some of the interesting observations are shown in Figure 1, where that of d(A1A2C3C4A5A6A7G8) is compared with those of oligomers derived by G base replacement at position 2 and T base substitutions at positions 2, 3, 4 and 8. Except for an enhancement in d(AGCCAAAG), reduced binding affinities are seen for all other substitutions. The greatly reduced binding affinity of d(ATCCAAAG) and the enhanced affinity of d(AGCCAAAG) demonstrate the crucial role played by A2 and the importance of having a purine base at this position. The relative importance of C3 versus C4 is nicely illustrated by the considerably greater reduction in binding affinity when C4 is replaced by a T base. Finally, the vital role played by G8 is readily apparent from the negligible affinity for ACTD of d(AACCAAAT).
CD and fluorescence spectral characteristics
CD difference spectra (drug/DNA – DNA) for d(AACCAAAG) and selective oligomers derived by base replacement are shown in Figure 2. Comparisons with T base substitutions at positions 2, 3 and 4 are made in Figure 2A. ACTD binding induces a strong negative CD maximum at 268 nm flanked by moderately positive maxima near 293 and 245 nm. T base replacement of A2 or C4 greatly reduces the CD intensity and is consistent with the weak binding affinity exhibited by d(ATCCAAAG) and d(AACTAAAG). Most interestingly, the induced CD spectrum upon ACTD binding to d(AATCAAAG) is nearly identical to that of d(AACCAAAG). This clearly suggests structural similarity between these two drug complexes, confirming a much more crucial role being played by C4 than by C3. Induced CD spectra on ACTD binding to d(AACCAAAG), d(AGCCAAAG) and oligomers derived by T base substitution at C3 or C4 of the latter are compared in Figure 2B. Dramatic spectral alterations are seen upon G base substitution at A2, which resulted in the appearance of a strong negative CD near 245 nm with concomitant intensity reduction and enhancement at 268 and 293 nm, respectively. Again, the near identical induced CD spectra of ACTD binding to d(AGCCAAAG) and d(AGTCAAAG) along with the diminished CD intensity on binding to d(AGCTAAAG), especially that at 268 nm, testify to the critical role of C4 in the binding process.
Figure 2. CD difference spectra (ACTD/DNA – DNA) for ACTD binding to d(AACCAAAG) and selective oligomers. (A) Comparison with T base substituted oligomers at positions 2, 3 and 4. (B) Comparison with d(AGCCAAAG) and its T base substituted oligomers at positions 3 and 4. CD measurements were made at room temperature using cylindrical cells of 2 cm path length. Concentrations for ACTD and DNA are 7 and 40 μM (in nucleotides), respectively.
The fluorescence intensity of 7-AM-ACTD (a fluorescent analog of ACTD) near 600 nm is enhanced upon binding to d(AACCAXYG). Thus, excitation difference spectra (drug/DNA – drug) via fluorescence monitoring at 600 nm for some selective oligomers are compared in Figure 3. The effects of T base substitutions at positions of 2, 3 and 4 of d(AACCAAAG) are shown in Figure 3A. The comparable intensities for d(AACCAAAG) and d(AATCAAAG) versus considerable diminution for d(AACTAAAG) support the more crucial role of C4 as compared to C3. This is further confirmed by the fluorescence intensity comparison of d(AGCCAAAG), d(AGTCAAAG) and d(AGCTAAAG), as shown in Figure 3B. The slightly higher intensity of d(AGCCAAAG) as compared to d(AACCAAAG) is consistent with the somewhat stronger ACTD binding affinity of the former, as revealed by equilibrium binding studies.
Figure 3. Fluorescence difference excitation spectra (drug/DNA – drug) at room temperature for 7AM-ACTD binding to d(AACCAAAG) and selective oligomers. (A) Comparison with T base substituted oligomers at positions 2, 3 and 4. (B) Comparison with d(AGCCAAAG) and its T base substituted oligomers at positions 3 and 4. Concentrations for drug and DNA are 2 and 40 μM. respectively. Fluorescence emission monitored at 600 nm.
DISCUSSION
This work was motivated by the earlier study of Yoo and Rill (16) in which they found d(AACCATAG) to exhibit a high affinity for ACTD and intimated that a TAG sequence at the 3'-end is essential for the strong binding. In an effort to elucidate the nature of such a binding and to verify their assertion on the TAG sequence requirement, systematic binding studies were carried out with oligomers of sequence motifs d(AACCAXYG). It was found that oligomers with XAG and XCG at the 3'-end, where X can be any base, all exhibit strong ACTD affinity. Although oligomers ending with XTG and XGG exhibit somewhat weaker binding, their strengths are still well in the μM–1 binding range.
To delineate the nature of the binding modes for these sequence motifs, systematic binding studies via base substitutions were made with d(A1A2C3C4A5A6A7G8), a sequence exhibiting a binding affinity comparable to that of d(AACCATAG) studied earlier. It was found that the 3'-terminus G base is indispensable for the strong ACTD binding of these oligomers. Although the 5'-terminus A can be replaced by any other base without significantly altering the ACTD binding strength, its removal greatly diminishes the binding affinity. This terminal base likely provides anchorage support for one of the pentapeptide rings of the drug. The two C bases are also important, with C4 appearing to be several-fold more crucial than C3. Interestingly, replacing A2 by a pyrimidine greatly diminishes the affinity of the oligomer for ACTD, whereas replacement by a G base enhances it considerably. Similar binding behaviors were also found for d(AACCATAG) and its related oligomers, although the data were not presented.
Elucidation of the nature of the strong ACTD binding to d(AACCAXYG) was greatly aided by the enhanced ACTD binding exhibited by d(AGCCAXYG). Thus, further studies were carried out with this and related sequences. Binding studies on oligomers obtained via base substitutions in d(AGCCAAAG) were made and the binding trends were found to be very similar to their d(AACCAAAG) counterparts. Worthy of mention is the fact that d(AGXCAAAG) (X C) still retain considerable ACTD binding affinity, whereas those of d(AGCXAAAG) (X C) are several-fold weaker, implying the greater role played by C4 as compared to C3. A binding model based on the terminal G folding back to base pair with the C bases was then proposed to account for these observations. Two binding modes coexist in such a model, with the stronger binding one corresponding to the terminal G folding back to base pair with C4 and with C3 looped out to make room for the ACTD chromophore to be stacked with G bases on both sides. The minor complex is formed by G folding back to base pair with C3 with the chromophore inserting at the GpC site without base displacement. This seems somewhat counter-intuitive, as one would expect the C3 base pairing conformation to be dominant because it would enable ACTD to bind at the classic GpC site. However, our previous NMR studies on ACTD binding to d(GGC-ACC-GCC), a hairpin with a 5'-GGC/CCG-5' stem, revealed that the dominant binding mode is for the chromophore to disrupt and loop out the central G·C base pair while intercalation at the classic GpC site turned out to form only a minor component. The extra stability of the base displaced mode derives from ACTD interactions with the looped out bases (19). The predominance of G base pairing with C4 over C3 is nicely supported by the comparable induced CD and fluorescence spectra of d(AGCCAAAG) and d(AGTCAAAG) and the much reduced intensity for d(AGCTAAAG) upon drug binding.
Proposed binding model for d(AACCAXYG)
Patterned after the binding model of d(AGCCAAAG), the modes of ACTD binding to d(AACCAAAG) (see Fig. 4), and by extension to d(AACCAXYG), now appear to be straightforward. The strong binding mode is for the 3'-terminal G base to base pair with C4, with the phenoxazone plane inserted at A2C3C4 by displacing the C3 base while opposite sides of the chromophore are stacked by A and G bases, respectively. The weaker binding mode should correspond to the G base pairing with C3 and similar A/G stacking without base displacement. These assertions are consistent with our observed ACTD binding affinities and the drug-induced CD as well as fluorescence characteristics of the related oligomers. It may also be worthy of mention that the trends and variations in ACTD binding observed with oligomers of d(AACCAXYG) (see Table 1) most likely resulted in large measure from how an AXY loop facilitates hairpin formation. It may be that AXA (23) and AXC (24) mini-loops facilitate better hairpin formation than AXT and AXG, so that oligomers ending with XAG and XCG are better ACTD binders than those ending with XTG and XGG at the 3'-side.
Figure 4. Schematic drawings of the ACTD binding model proposed for d(AACCAAAG). The rectangular box represents the phenoxazone chromophore of ACTD.
The complexation of ACTD to DNA involves base stacking, hydrogen bonding and hydrophobic forces. Assuming comparable stacking interactions between the A/chromophore/G stack and the G/chromophore/G stack, the reduced ACTD affinity for d(AACCAAAG) as compared to d(AGCCAAAG) most likely resulted from one less hydrogen bond between the base and the cyclo-pentapeptide ring (one AN3–ThrHN hydrogen bond is expected for the A/chromophore stack, while one GN3–ThrHN hydrogen bond and one G2NH2–Thr(C=O) hydrogen bond are expected for the G/chromophore stack). A more general binding principle for ACTD can thus be stated as: the phenoxazone prefers to be stacked on both sides by purine bases, with G/G favored over A/G. Nonetheless, the novel A/chromophore/G stack appears to be strong enough in some related oligomers and should be amenable to NMR studies, which we are currently pursuing. It should be noted that the relative importance of stacking interactions between adenine versus guanine with the phenoxazone chromophore had earlier been investigated by Krugh et al. (25) using dideoxynucleotides.
The structures of an 11mer d(G1T2T3A4A5C6C7 A8T9A10G11) and its ACTD complex had earlier been investigated by Su (21) using 2-dimensional 1H NMR (in a 500 MHz instrument) and molecular modeling. The oligomer was shown to adopt a stable hairpin structure with an ACCA loop. The hairpin stem consists of two Watson–Crick A:T base pairs, a G:T mismatch pair and an unpaired 5'-terminal G. Their NMR data on the drug complex were interpreted in terms of intercalation of the drug at the T2:G11–T3:A10 step. This conclusion is somewhat surprising based on the elucidated G or A/chromophore/G binding principle described in the present manuscript. With the suggested hairpin of the ACCA loop it would have been expected that ACTD binds at the G1–T2:G11 step to achieve G/chromophore/G stacking or, analogous to what is described in this report, that the 3'-end G11 folds back to form a Watson–Crick pair with C7 and a hairpin with an ATA mini-loop. The ACTD chromophore then stacks between A5 and G11, with a looped out C6 residue. The 5'-GTT nucleotides are not necessary for complex stabilization, since the cyclo-pentapeptide lactone rings of ACTD only span 2 nt (26). A more detailed NMR investigation at higher field strength may be warranted to resolve this quandary.
Binding of ACTD to single-stranded DNA has been implicated in biological activities, including inhibition of viral ligase (27), helicase (28) and (–) strand transfer by HIV reverse transcriptases (29–32). Understanding ACTD binding to sequences which do not contain classic GpC sites should provide considerable insights into its mode of binding to DNA and assist in uncovering other new strong binding sequences. Furthermore, studying ligands that bind to single-stranded DNA with particular sequence preferences may be of importance in the regulation of processes that require single-stranded DNA as an intermediate. The affinity of ACTD for some hairpins raises the possibility that the drug could inhibit transcription by stabilizing cruciforms extruded from the transcription bubble. Stabilization by ACTD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ACTD or other related compounds may serve as a sequence-specific ssDNA binding agent that inhibits HIV and other retroviruses that replicate via ssDNA intermediates. For this aim to be realized, it must be demonstrated that the observed strong ACTD binding reported here is not merely the consequence of end effects. Although no systematic studies have been carried out for this purpose, equilibrium binding measurements were made with d(AACCAAAGX) where X = A, T, G or C, resulting in binding constants ranging from 1.2 x 106 for X = T to 11 x 106 M–1 for X = C. More studies are needed to draw any firm conclusions on the end-effect.
REFERENCES
Goldberg,I.H. and Friedman,P.A. (1971) Antibiotics and nucleic acids. Annu. Rev. Biochem., 40, 775–810.
Muller,W. and Crother,D.M. (1968) Studies of the binding of actinomycin and related compounds to DNA. J. Mol. Biol., 35, 251–290.
Waring,M.J. (1981) DNA modification and cancer. Annu. Rev. Biochem., 50, 159–192.
Aivasashvilli,V.A. and Beabealashvilli,R.S. (1983) Sequence-specific inhibition of RNA elogation by actinomycin D. FEBS Lett., 160, 124–128.
Phillips,D.R. and Crothers,D.M. (1986) Kinetics and sequence specificity of drug-DNA interactions: an in vitro transcription assay. Biochemistry, 25, 7355–7362.
Sobell,H.M. and Jain,S.C. (1972) Stereochemistry of actinomycin binding to DNA II. Detailed molecular model of actinomycin–DNA complex and its implication. J. Mol. Biol., 68, 21–34.
Kamitori,S. and Takusagawa,F. (1992) Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D. J. Mol. Biol., 225, 445–456.
Snyder,J.G., Hartman,N.G., D’Estantoit,B.L., Kennard,O., Remeta,D.P. and Breslauer,K.P. (1989) Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites. Proc. Natl Acad. Sci. USA, 86, 3968–3972.
Rill,R.L., Marsch,G.A. and Graves,D.E. (1989) 7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D. J. Biomol. Struct. Dyn., 7, 591–605.
Bailey,S.A., Graves,D.E. and Rill,R. (1994) Binding of actinomycin D to the T(G)nT motif of double-stranded DNA: determination of the guanine requirement in nonclassical, non-GpC binding sites. Biochemistry, 33, 11493–11500.
Wadkins,R.M. and Jovin,T.M. (1991) Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA. Biochemistry, 30, 9469–9478.
Hsieh,Y.L., Li,Y.T. and Henion,J.D. (1994) Studies of non-covalent interactions of actinomycin D with single-stranded oligodeoxynucleotides by ion spray mass spectrometry and tandem mass spectrometry. Biol. Mass Spectrom., 116, 272–276.
Wadkins,R.M., Jares-Erijman,E.A., Klement,R., Rudiger,A. and Jovin,T.M. (1996) Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and 1H NMR spectroscopy. J. Mol. Biol., 262, 53–68.
Rill,R.L. and Hecker,K.H. (1996) Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptases and other polymerases. Biochemistry, 35, 3525–3533.
Wadkins,R.M., Vladu,B. and Tung,C.-S. (1998) Actinomycin D binds to metastable hairpins in single-stranded DNA. Biochemistry, 37, 11915–11923.
Yoo,H. and Rill,R.L. (2001) Actinomycin D binding to unstructured, single-stranded DNA. J. Mol. Recognit., 14, 145–150.
Chen,F.-M. and Sha,F. (2001) Actinomycin D binds strongly to d(TGTCATTG), a single-stranded DNA devoid of GpC sites. Biochemistry, 40, 5218–5225.
Chen,F.-M., Sha,F., Chin,K.-H. and Chou,S.-H. (2003) Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCTnG) and d(TGTnGTCT). Biophys. J., 84, 432–439.
Chou,S.-H., Chin,K.-H. and Chen,F.-M. (2002) Looped-out and perpendicular: deformation of Watson-Crick base pair associated with actinomycin D binding. Proc. Natl Acad. Sci. USA, 99, 6625–6630.
Chin,K.-H., Chen,F.M. and Chou,S.-H. (2003) Solution structure of actinomycin D/5'-CCGTTTTGTGG-3' complex: drug interaction with tandem G·T mismatches and hairpin loop backbone. Nucleic Acids Res., 31, 2622–2629.
Su,L. (1995) Conformations of single stranded DNA oligonucleotides and their actinomycin D complexes studies by high resolution 1H NMR spectroscopy. PhD dissertation, Florida State University, Tallahassee, FL.
Fasman,G.D. (ed.) (1975) CRC Handbook of Biochemistry and Molecular Biology, 3rd Edn. Chemical Rubber Publishing Co., Cleveland, OH, Vol. I, p. 589.
Chou,S.-H., Zhu,L., Gao,Z., Cheng,J.-W. and Reid,B.R. (1996) Hairpin loops consisting of single adenine residues closed by sheared A:A and G:G pairs formed by the DNA triplets AAA and GAG: solution structure of the d(GTACAAAGTAC) hairpin. J. Mol. Biol., 264, 981–1001.
Chou,S.-H., Tseng,Y.-Y. and Wang,S.-W. (1999) Stable sheared A:C pair in DNA hairpins. J. Mol. Biol., 287, 301–313.
Krugh,T.R., Mooberry,E.S. and Chiao,Y.-C.C. (1977) Proton magnetic resonance studies of actinomycin D complexes with mixtures of nucleotides as models for the binding of drug to DNA. Biochemistry, 16, 740–747.
Liu,X., Chen,H. and Patel,D.J. (1991) Solution structure of actinomycin–DNA complexes: drug intercalation at isolated G-C sites. J. Biomol. NMR, 1, 323–347.
Shuman,S. (1995) Vaccinia virus DNA ligase: specificity, fidelity and inhibition. Biochemistry, 34, 16138–16147.
Tuteja,N., Phan,T.N., Tuteja,R., Ochem,A. and Falaschi,A. (1997) Inhibition of DNA unwinding and ATPase activities of human DNA helicase II by chemotherapeutic agents. Biochem. Biophys. Res. Commun., 236, 636–640.
Davis,W.R., Gabbara,S., Hupe,D. and Peliska,J.A. (1998) Actinomycin D inhibition of DNA strand transfer reaction catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein. Biochemistry, 37, 14213–14221.
Guo,J., Wu,T., Bes,J., Henderson,L.E. and Levin,J.G. (1988) Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays. J. Virol., 72, 6716–6724.
Jeeninga,R.E., Huthoff,H.T., Gultyaev,A.P. and Berkhout,B. (1998) The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription. Nucleic Acids Res., 26, 5472–5479.
Gabbara,S., Davis,W.R., Hupe,L. and Peliska,J.A. (1999) Inhibitors of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Biochemistry, 38, 13070–13076.(Fu-Ming Chen*, Feng Sha, Ko-Hsin Chin1 a)
*To whom correspondence should be addressed. Tel: +1 615 963 5325; Fax: +1 615 963 5434; Email: fchen@tnstate.edu
ABSTRACT
Earlier studies by others had indicated that actinomycin D (ACTD) binds well to d(AACCATAG) and the end sequence TAG-3' is essential for its strong binding. In an effort to verify these assertions and to uncover other possible strong ACTD binding sequences as well as to elucidate the nature of their binding, systematic studies have been carried out with oligomers of d(AACCAXYG) sequence motifs, where X and Y can be any DNA base. The results indicate that in addition to TAG-3', oligomers ending with XAG-3' and XCG-3' all provide binding constants 1 x 107 M–1 and even sequences ending with XTG-3' and XGG-3' exhibit binding affinities in the range 1–8 x 106 M–1. The nature of the strong ACTD affinity of the sequences d(A1A2C3C4A5X6Y7G8) was delineated via comparative binding studies of d(AACCAAAG), d(AGCCAAAG) and their base substituted derivatives. Two binding modes are proposed to coexist, with the major component consisting of the 3'-terminus G base folding back to base pair with C4 and the ACTD inserting at A2C3C4 by looping out the C3 while both faces of the chromophore are stacked by A and G bases, respectively. The minor mode is for the G to base pair with C3 and to have the same A/chromophore/G stacking but without a looped out base. These assertions are supported by induced circular dichroic and fluorescence spectral measurements.
INTRODUCTION
Actinomycin D (ACTD) is an anticancer antibiotic best known for inhibiting transcription by binding to double-stranded DNA (1–5). It is a chromopeptide consisting of a phenoxazinone planar chromophore with two pentapeptide rings attached. Its mode of DNA binding has been shown to be intercalative, in which the chromophore inserts in between the DNA base pairs while the two chains of the pentapeptide rest in the minor groove. The GpC sequence specificity of this drug has been well characterized and originates from its ability to form two hydrogen bonds between each guanine and a pentapeptide ring (6,7).
However, there have been reports to indicate that this drug can also bind strongly to some non-GpC-containing sequences (8–10) as well as to some single-stranded (ss)DNA (11–16). Studies in our laboratory have discovered that ACTD binds strongly to the sequence d(TGTCATTG) of apparent single-stranded conformation and without GpC sites (17). A fold-back binding model was speculated wherein the planar phenoxazone inserts at the GTC site with a looped out T base whereas the 3'-terminus G folds back to form a base pair with the internal C and stacks on the opposite face of the chromophore. Additional supports for the hairpin binding model came from systematic binding studies with oligomers of sequence motifs d(TGTCTnG) and d(TGTnGTCT) that are devoid of GpC sequences and exist predominantly in single-stranded conformation (18).
Our recent NMR studies on the complex structure of ACTD bound to hairpins containing the DNA recognition site 5'-GXC/CYG-5' in the duplex stem (where X/Y is a G·C or T·A Watson–Crick base pair) further revealed a rather unusual interaction mode for the intercalated drug, in which the central Watson–Crick X/Y base pairs are disrupted and displaced by the ACTD chromophore (19). The looped out bases are not disordered but interact perpendicularly with the base/chromophore and form specific hydrogen bonds with DNA. These results support the binding principle that the ACTD chromophore prefers to be stacked on each side by the 3'-end of the G and demonstrate the important roles played by the looped out bases. Based on such a principle, a strong ACTD binding sequence of seemingly ssDNA oligomer 5'-CCGTT3GTGG-3' was designed and its high quality NMR spectra allowed us to determine its ACTD complex structure (20). The DNA oligomer 5'-CCGTT3GTGG-3' forms a hairpin structure in the complex, with tandem G·T mismatches in the stem region next to a loop of three stacked thymine bases pointing towards the major groove. Bipartite hydrogen bonds were detected for the G·T mismatches that further stabilize this unusual DNA hairpin. The phenoxazone chromophore of ACTD intercalates nicely between the tandem G·T mismatches in essentially one major orientation. The hydrophobic G/phenoxazone/G interaction in the ACTD/5'-CCGTT3GTGG-3' complex was found to be more robust than that of the classical ACTD/5'-GpC-3' complex, consistent with the roughly 2-fold stronger binding of ACTD with 5'-CCGTT3GTGG-3' than with its 5'-CCGCT3GCGG-3' counterpart.
Although other hairpin models for single-stranded ACTD binding had earlier been suggested, the sequence context appeared to be quite distinct (15,16,21). Systematic binding studies of 7-amino-actinomycin D (7-AM-ACTD) with single-stranded oligomers containing -TAGT- sequences led to the suggestion that the drug binds to metastable hairpins. The hairpins bound most tightly by ACTD appear to contain non-Watson–Crick base pairs, including an A/G and two T/T mismatches (15). Studies by Rill’s group, however, suggested that binding of ACTD to ssDNA might not necessarily require DNA secondary structure at the outset (16,21). Oligonucleotide d(GTTAACCATAG) was identified as one of the rare ssDNAs that binds ACTD with high affinity. Although this sequence does not appear a priori favorable for haripin formation, NMR and other studies have suggested that in the ACTD complex the oligonucleotide adopts a stable, folded hairpin with an ACCA loop and 5'-TTA/GAT-5' stem (21). A more systematic binding study (16) was then carried out on a series of oligonucleotides corres ponding to progressive shortening of d(GTTAACCATAG) from the 5'-end plus a large number of related sequences. A minimum sequence of d(AACCATAG) was found sufficient for tight binding and indeed exhibits the highest ACTD affinity among the oligomers studied. It was also concluded that a TAG-3' terminal sequence is essential for strong ACTD binding. These results support the hypothesis that the initial binding of ACTD to certain ssDNA sequences can induce a substantial conformational change in the flanking sequences to achieve a highly stable ssDNA/ACTD complex and show that mechanisms of ACTD binding to DNA are more diverse than commonly perceived.
Elucidation on the nature of ACTD binding to d(AACCATAG) is important and could provide additional insight into the action of this drug, since it is devoid of G/chromophore/G stacking in complex formation. To this end, our laboratory has now carried out systematic ACTD binding studies with d(AACCAXYG) and related sequences. The results are presented herein and a binding model is proposed to account for the observed binding behaviors.
MATERIALS AND METHODS
Synthetic oligonucleotides were purchased from ResGen (Huntsville, AL) and used without further purification. These oligomers were purified by the vendor using reverse phase oligonucleotide purification cartridges and exhibited single band electrophoretic mobilities in denaturing polyacrylamide gel electrophoresis with stated purity of 95%. Concentrations of the DNA solutions (in nucleotides) were determined by measuring the absorbances at 260 nm after melting (at 95°C). The extinction coefficients of DNA oligomers were obtained via nearest neighbor approximation using mono- and dinucleotide values tabulated in Fasman (22). ACTD and 7-AM-ACTD were purchased from Serva. Concentrations of the drug solutions were determined by measuring the absorbances at 440 nm (for ACTD) and 528 nm (for 7-AM-ACTD), using extinction coefficients of 24 500 and 23 600 cm–1 M–1, respectively. Stock solutions of oligonucleotides and drugs were prepared by dissolution in 10 mM Tris–borate buffer pH 8 containing 0.1 M NaCl and 1 mM MgCl2. Absorption spectra were measured with a Cary 1E spectrophotometric system. Absorption spectral titrations were carried out starting with 2 ml of a 5 μM ACTD solution followed by progressive additions of the oligomer stock at equal time intervals. Absorbance differences between 427 and 480 nm during absorption spectral titrations were used to obtain the binding isotherms. Fluorescence spectra were obtained with a SLM 48000S system at room temperature using a cell of 1 cm path length. Circular dichroic (CD) spectra were measured at room temperature with a Jasco J-500A recording spectropolarimeter using water-jacketed cylindrical cells of 2 cm path length.
Association binding constants (K) were extracted via non-linear least squares fits on the experimental binding isotherms using a 1:1 drug to strand binding model: D + S = DS, where D, S and DS are free drug, free single-stranded DNA and drug–DNA complex, respectively. By means of equations for the mass balances of drug and DNA (in strand), the following equations can be derived:
D2K + D = Dt
S = Dt(X – 1) + D
Y = (D + 1 K S)D/Dt
where X St/Dt, D and 1 are the extinction coefficients of the free and bound drugs and Dt and St are the total drug and DNA strand concentrations, respectively, at each point of the titration. Experimental binding isotherms were plotted as the apparent extinction coefficient (Y) versus X and non-linear least squares fits to the above equations were made by initially fixing the known D and subsequently relaxing it to obtain the best fit to extract the binding constant K. The non-linear least squares fit program in Micromath (Salt Lake City, UT) was used for our fitting purpose.
RESULTS
Is the TAG sequence at the 3'-end of d(AACCATAG) essential for its strong ACTD binding?
In an effort to ascertain the validity of the assertion of a TAG-3' requirement in the strong ACTD binding of d(AACCATAG), systematic ACTD binding studies were made with oligonucleotides with d(AACCAXYG) sequence motifs, where X and Y can be any base. The results are shown in Table 1. These binding constants were extracted via non-linear least squares fits using a 1:1 drug to strand binding model on the experimental data points. Some representative binding isotherms and the goodness of fit can be seen in Figure 1, with their significance to be discussed later. It is found that in addition to TAG, sequences such as AAG, GAG, and CAG, as well as ACG, TCG, GCG and CCG, at the 3'-end all provide very strong ACTD binding, exhibiting binding constants 1 x 107 M–1. However, significant ACTD binding affinities are also exhibited by the rest of the sequences, with binding constants ranging from 1 to 8 x 106 M–1. These results suggest that the trinucleotide sequences XYG at the 3'-end can provide strong ACTD binding if preceded by AACCA at the 5'-end. It is noteworthy that oligomers with XAG and XCG endings exhibit decidedly stronger ACTD affinities than the corresponding XTG- or XGG-containing sequences.
Table 1. Comparison of ACTD binding constants at 25°C for the d(AACCAXYG) oligomers
Figure 1. Equilibrium binding isotherms for ACTD binding to d(AACCAAAG) and selective oligomers derived by single base substitution. Absorbance spectral titrations were carried out at 25°C and the absorbance difference A is that between 427 and 480 nm. Concentrations of both ACTD and DNA (in strands) are expressed in μM. Solid curves are those of non-linear least squares fits of the experimental isotherms using the 1:1 drug to strand binding model.
The G base at the 3'-end is vital for strong ACTD binding
To elucidate the nature of and to uncover the crucial DNA bases responsible for the strong ACTD binding of these oligomers, further studies were made with related oligonucleotides via base substitutions in d(AACCAAAG), a sequence which is seen to bind as strongly to ACTD as d(AACCATAG) and exhibits a binding constant of 10 x 106 M–1 (see Table 1). The most obvious starting point is to find out if a G base in the sequence is essential for binding, as it has the capacity to form hydrogen bonds with the drug threonine bases in the pentapeptide rings. Thus, oligomers with the 3'-terminal G replaced by A, T or C were studied and the results indicate that d(AACCAAAA), d(AACCAAAT) and d(AACCAAAC) all exhibited binding constants <<1 x 105 M–1, at least two orders of magnitude weaker than the parent oligomer containing the G base (see Table 2). These results indicate that the G base at the 3'-end is vital for the strong ACTD binding observed with these oligomers.
Table 2. Comparison of ACTD binding constants at 25°C for some oligomers derived from base substitutions of d(AACCAAAG)
Both C bases in AACC are essential but C4 is significantly more important than C3
To assess the contributions made by the two C bases as well as to compare their relative importance, ACTD binding studies were carried out with base replacement of either of the C residues in d(A1A2C3C4A5A6A7G8) and the results are also included in Table 2. Binding constants of 0.64, 1.2 and 0.71 x 106 M–1 were found for d(AAACAAAG), d(AATCAAAG) and d(AAGCAAAG), respectively, whereas values of 0.11, 0.15 and 0.18 x 106 M–1 were found for d(AACAAAAG), d(AACTAAAG) and d(AACGAAAG), respectively. Although considerable reductions in binding affinity are observed for base substitutions at either position, replacements of the C4 base are seen to be decidedly more affective. This suggests that both C bases contribute substantially, but that C4 appears to make a considerably (several-fold) more important contribution to the strong ACTD binding of these oligomers.
Replacement of the A base at the 5'-terminus does not greatly affect binding but its removal does
The roles played by the two A bases at the 5'-end were assessed by binding studies with base substitution of either of them and the results are also shown in Table 2. ACTD binding constants for d(TACCAAAG), d(GACCAAAG) d(CACCAAAG) were found to be 7.8, 15 and 4.8 x 106 M–1, respectively. Thus, base replacement of the terminal A does not greatly affect the binding strength. On the other hand, removal of the 5'-terminal base resulting in the heptamer d(ACCAAAG) diminishes the binding affinity by 20-fold.
Replacement of A2 by pyrimidines greatly diminishes while replacement with G base significantly enhances ACTD binding
A very different trend is observed when A2 is replaced by another DNA base. ACTD binding constants for d(ATCCAAAG), d(AGCCAAAG) and d(ACCCAAAG) were found to be 0.3, 100 and 0.4 x 106 M–1, respectively. Thus, substitution with a pyrimidine base T or C resulted in more than an order of magnitude reduction in the binding affinity. In contrast, a G base substitution resulted in a considerably higher binding strength. Such an enhancement upon G base substitution, coupled with the diminution of binding affinity upon pyrimidine substitution, provide us with considerable insights as to the possible modes of ACTD binding to the d(AACCAXYG) oligomers.
Binding studies with oligomers derived from base substitution in d(AGCCAAAG)
The enhanced ACTD binding of d(AGCCAAAG) provides us with a good road map for delineating the nature of ACTD binding to d(AACCAAAG), as the binding mode of the former can more easily be deduced due to the presence of an extra G at a strategic position in the sequence for possible utilization of the G/chromophore/G binding principle. To this end, ACTD binding studies were made with oligomers derived by base replacement in d(AGCCAAAG) and some of the results are shown in Table 3 (results on base replacement in A5A6A7 are not included as no dramatic variation was observed). The critical role played by the 3'-terminus G base is again illustrated by the much-weakened ACTD binding affinity when this base is replaced. Despite the presence of GpC sites, ACTD binding constants for d(AGCCAAAA), d(AGCCAAAT) and d(AGCCAAAC) were found to be 0.05, 0.08 and 0.07 x 106 M–1, respectively. These values are more than three orders of magnitude weaker than the parent octamer d(AGCCAAAG). In contrast, base replacement of the 5'-terminus A base appears to have minimal effect on the affinity for ACTD, as demonstrated by the high binding constants of 70, 100 and 36 x 106 M–1 retained by d(TGCCAAAG), d(GGCCAAAG) and d(CGCCAAAG), respectively. Base replacement of C3 (so that only C4 can form a base pair with G) resulted in d(AGACAAAG), d(AGTCAAAG) and d(AGGCAAAG), which exhibit ACTD binding affinities of 15, 8 and 10 x 106 M–1, respectively. This is still very strong binding, despite the fact that they are about an order of magnitude weaker than the 100 x 106 M–1 exhibited by the parent oligomer d(AGCCAAAG). In fact they are several-fold stronger than those obtained via base replacement at C4 (so that only C3 can now form a base pair with G) to result in d(AGCAAAAG), d(AGCTAAAG) and d(AGCGAAAG), which exhibit binding constants of 0.6, 1.8 and 2.4 x 106 M–1, respectively. These results suggest that C4 plays a more pivotal role than C3 during ACTD binding, a result akin to what was found for the d(AACCAAAG) counterparts.
Table 3. Comparison of ACTD binding affinities at 25°C for some oligomers derived from base substitutions of d(AGCCAAAG).
Representative binding isotherms
Selective ACTD binding isotherms illustrating some of the interesting observations are shown in Figure 1, where that of d(A1A2C3C4A5A6A7G8) is compared with those of oligomers derived by G base replacement at position 2 and T base substitutions at positions 2, 3, 4 and 8. Except for an enhancement in d(AGCCAAAG), reduced binding affinities are seen for all other substitutions. The greatly reduced binding affinity of d(ATCCAAAG) and the enhanced affinity of d(AGCCAAAG) demonstrate the crucial role played by A2 and the importance of having a purine base at this position. The relative importance of C3 versus C4 is nicely illustrated by the considerably greater reduction in binding affinity when C4 is replaced by a T base. Finally, the vital role played by G8 is readily apparent from the negligible affinity for ACTD of d(AACCAAAT).
CD and fluorescence spectral characteristics
CD difference spectra (drug/DNA – DNA) for d(AACCAAAG) and selective oligomers derived by base replacement are shown in Figure 2. Comparisons with T base substitutions at positions 2, 3 and 4 are made in Figure 2A. ACTD binding induces a strong negative CD maximum at 268 nm flanked by moderately positive maxima near 293 and 245 nm. T base replacement of A2 or C4 greatly reduces the CD intensity and is consistent with the weak binding affinity exhibited by d(ATCCAAAG) and d(AACTAAAG). Most interestingly, the induced CD spectrum upon ACTD binding to d(AATCAAAG) is nearly identical to that of d(AACCAAAG). This clearly suggests structural similarity between these two drug complexes, confirming a much more crucial role being played by C4 than by C3. Induced CD spectra on ACTD binding to d(AACCAAAG), d(AGCCAAAG) and oligomers derived by T base substitution at C3 or C4 of the latter are compared in Figure 2B. Dramatic spectral alterations are seen upon G base substitution at A2, which resulted in the appearance of a strong negative CD near 245 nm with concomitant intensity reduction and enhancement at 268 and 293 nm, respectively. Again, the near identical induced CD spectra of ACTD binding to d(AGCCAAAG) and d(AGTCAAAG) along with the diminished CD intensity on binding to d(AGCTAAAG), especially that at 268 nm, testify to the critical role of C4 in the binding process.
Figure 2. CD difference spectra (ACTD/DNA – DNA) for ACTD binding to d(AACCAAAG) and selective oligomers. (A) Comparison with T base substituted oligomers at positions 2, 3 and 4. (B) Comparison with d(AGCCAAAG) and its T base substituted oligomers at positions 3 and 4. CD measurements were made at room temperature using cylindrical cells of 2 cm path length. Concentrations for ACTD and DNA are 7 and 40 μM (in nucleotides), respectively.
The fluorescence intensity of 7-AM-ACTD (a fluorescent analog of ACTD) near 600 nm is enhanced upon binding to d(AACCAXYG). Thus, excitation difference spectra (drug/DNA – drug) via fluorescence monitoring at 600 nm for some selective oligomers are compared in Figure 3. The effects of T base substitutions at positions of 2, 3 and 4 of d(AACCAAAG) are shown in Figure 3A. The comparable intensities for d(AACCAAAG) and d(AATCAAAG) versus considerable diminution for d(AACTAAAG) support the more crucial role of C4 as compared to C3. This is further confirmed by the fluorescence intensity comparison of d(AGCCAAAG), d(AGTCAAAG) and d(AGCTAAAG), as shown in Figure 3B. The slightly higher intensity of d(AGCCAAAG) as compared to d(AACCAAAG) is consistent with the somewhat stronger ACTD binding affinity of the former, as revealed by equilibrium binding studies.
Figure 3. Fluorescence difference excitation spectra (drug/DNA – drug) at room temperature for 7AM-ACTD binding to d(AACCAAAG) and selective oligomers. (A) Comparison with T base substituted oligomers at positions 2, 3 and 4. (B) Comparison with d(AGCCAAAG) and its T base substituted oligomers at positions 3 and 4. Concentrations for drug and DNA are 2 and 40 μM. respectively. Fluorescence emission monitored at 600 nm.
DISCUSSION
This work was motivated by the earlier study of Yoo and Rill (16) in which they found d(AACCATAG) to exhibit a high affinity for ACTD and intimated that a TAG sequence at the 3'-end is essential for the strong binding. In an effort to elucidate the nature of such a binding and to verify their assertion on the TAG sequence requirement, systematic binding studies were carried out with oligomers of sequence motifs d(AACCAXYG). It was found that oligomers with XAG and XCG at the 3'-end, where X can be any base, all exhibit strong ACTD affinity. Although oligomers ending with XTG and XGG exhibit somewhat weaker binding, their strengths are still well in the μM–1 binding range.
To delineate the nature of the binding modes for these sequence motifs, systematic binding studies via base substitutions were made with d(A1A2C3C4A5A6A7G8), a sequence exhibiting a binding affinity comparable to that of d(AACCATAG) studied earlier. It was found that the 3'-terminus G base is indispensable for the strong ACTD binding of these oligomers. Although the 5'-terminus A can be replaced by any other base without significantly altering the ACTD binding strength, its removal greatly diminishes the binding affinity. This terminal base likely provides anchorage support for one of the pentapeptide rings of the drug. The two C bases are also important, with C4 appearing to be several-fold more crucial than C3. Interestingly, replacing A2 by a pyrimidine greatly diminishes the affinity of the oligomer for ACTD, whereas replacement by a G base enhances it considerably. Similar binding behaviors were also found for d(AACCATAG) and its related oligomers, although the data were not presented.
Elucidation of the nature of the strong ACTD binding to d(AACCAXYG) was greatly aided by the enhanced ACTD binding exhibited by d(AGCCAXYG). Thus, further studies were carried out with this and related sequences. Binding studies on oligomers obtained via base substitutions in d(AGCCAAAG) were made and the binding trends were found to be very similar to their d(AACCAAAG) counterparts. Worthy of mention is the fact that d(AGXCAAAG) (X C) still retain considerable ACTD binding affinity, whereas those of d(AGCXAAAG) (X C) are several-fold weaker, implying the greater role played by C4 as compared to C3. A binding model based on the terminal G folding back to base pair with the C bases was then proposed to account for these observations. Two binding modes coexist in such a model, with the stronger binding one corresponding to the terminal G folding back to base pair with C4 and with C3 looped out to make room for the ACTD chromophore to be stacked with G bases on both sides. The minor complex is formed by G folding back to base pair with C3 with the chromophore inserting at the GpC site without base displacement. This seems somewhat counter-intuitive, as one would expect the C3 base pairing conformation to be dominant because it would enable ACTD to bind at the classic GpC site. However, our previous NMR studies on ACTD binding to d(GGC-ACC-GCC), a hairpin with a 5'-GGC/CCG-5' stem, revealed that the dominant binding mode is for the chromophore to disrupt and loop out the central G·C base pair while intercalation at the classic GpC site turned out to form only a minor component. The extra stability of the base displaced mode derives from ACTD interactions with the looped out bases (19). The predominance of G base pairing with C4 over C3 is nicely supported by the comparable induced CD and fluorescence spectra of d(AGCCAAAG) and d(AGTCAAAG) and the much reduced intensity for d(AGCTAAAG) upon drug binding.
Proposed binding model for d(AACCAXYG)
Patterned after the binding model of d(AGCCAAAG), the modes of ACTD binding to d(AACCAAAG) (see Fig. 4), and by extension to d(AACCAXYG), now appear to be straightforward. The strong binding mode is for the 3'-terminal G base to base pair with C4, with the phenoxazone plane inserted at A2C3C4 by displacing the C3 base while opposite sides of the chromophore are stacked by A and G bases, respectively. The weaker binding mode should correspond to the G base pairing with C3 and similar A/G stacking without base displacement. These assertions are consistent with our observed ACTD binding affinities and the drug-induced CD as well as fluorescence characteristics of the related oligomers. It may also be worthy of mention that the trends and variations in ACTD binding observed with oligomers of d(AACCAXYG) (see Table 1) most likely resulted in large measure from how an AXY loop facilitates hairpin formation. It may be that AXA (23) and AXC (24) mini-loops facilitate better hairpin formation than AXT and AXG, so that oligomers ending with XAG and XCG are better ACTD binders than those ending with XTG and XGG at the 3'-side.
Figure 4. Schematic drawings of the ACTD binding model proposed for d(AACCAAAG). The rectangular box represents the phenoxazone chromophore of ACTD.
The complexation of ACTD to DNA involves base stacking, hydrogen bonding and hydrophobic forces. Assuming comparable stacking interactions between the A/chromophore/G stack and the G/chromophore/G stack, the reduced ACTD affinity for d(AACCAAAG) as compared to d(AGCCAAAG) most likely resulted from one less hydrogen bond between the base and the cyclo-pentapeptide ring (one AN3–ThrHN hydrogen bond is expected for the A/chromophore stack, while one GN3–ThrHN hydrogen bond and one G2NH2–Thr(C=O) hydrogen bond are expected for the G/chromophore stack). A more general binding principle for ACTD can thus be stated as: the phenoxazone prefers to be stacked on both sides by purine bases, with G/G favored over A/G. Nonetheless, the novel A/chromophore/G stack appears to be strong enough in some related oligomers and should be amenable to NMR studies, which we are currently pursuing. It should be noted that the relative importance of stacking interactions between adenine versus guanine with the phenoxazone chromophore had earlier been investigated by Krugh et al. (25) using dideoxynucleotides.
The structures of an 11mer d(G1T2T3A4A5C6C7 A8T9A10G11) and its ACTD complex had earlier been investigated by Su (21) using 2-dimensional 1H NMR (in a 500 MHz instrument) and molecular modeling. The oligomer was shown to adopt a stable hairpin structure with an ACCA loop. The hairpin stem consists of two Watson–Crick A:T base pairs, a G:T mismatch pair and an unpaired 5'-terminal G. Their NMR data on the drug complex were interpreted in terms of intercalation of the drug at the T2:G11–T3:A10 step. This conclusion is somewhat surprising based on the elucidated G or A/chromophore/G binding principle described in the present manuscript. With the suggested hairpin of the ACCA loop it would have been expected that ACTD binds at the G1–T2:G11 step to achieve G/chromophore/G stacking or, analogous to what is described in this report, that the 3'-end G11 folds back to form a Watson–Crick pair with C7 and a hairpin with an ATA mini-loop. The ACTD chromophore then stacks between A5 and G11, with a looped out C6 residue. The 5'-GTT nucleotides are not necessary for complex stabilization, since the cyclo-pentapeptide lactone rings of ACTD only span 2 nt (26). A more detailed NMR investigation at higher field strength may be warranted to resolve this quandary.
Binding of ACTD to single-stranded DNA has been implicated in biological activities, including inhibition of viral ligase (27), helicase (28) and (–) strand transfer by HIV reverse transcriptases (29–32). Understanding ACTD binding to sequences which do not contain classic GpC sites should provide considerable insights into its mode of binding to DNA and assist in uncovering other new strong binding sequences. Furthermore, studying ligands that bind to single-stranded DNA with particular sequence preferences may be of importance in the regulation of processes that require single-stranded DNA as an intermediate. The affinity of ACTD for some hairpins raises the possibility that the drug could inhibit transcription by stabilizing cruciforms extruded from the transcription bubble. Stabilization by ACTD of a hairpin containing non-canonical stem base pairs further strengthens the notion that ACTD or other related compounds may serve as a sequence-specific ssDNA binding agent that inhibits HIV and other retroviruses that replicate via ssDNA intermediates. For this aim to be realized, it must be demonstrated that the observed strong ACTD binding reported here is not merely the consequence of end effects. Although no systematic studies have been carried out for this purpose, equilibrium binding measurements were made with d(AACCAAAGX) where X = A, T, G or C, resulting in binding constants ranging from 1.2 x 106 for X = T to 11 x 106 M–1 for X = C. More studies are needed to draw any firm conclusions on the end-effect.
REFERENCES
Goldberg,I.H. and Friedman,P.A. (1971) Antibiotics and nucleic acids. Annu. Rev. Biochem., 40, 775–810.
Muller,W. and Crother,D.M. (1968) Studies of the binding of actinomycin and related compounds to DNA. J. Mol. Biol., 35, 251–290.
Waring,M.J. (1981) DNA modification and cancer. Annu. Rev. Biochem., 50, 159–192.
Aivasashvilli,V.A. and Beabealashvilli,R.S. (1983) Sequence-specific inhibition of RNA elogation by actinomycin D. FEBS Lett., 160, 124–128.
Phillips,D.R. and Crothers,D.M. (1986) Kinetics and sequence specificity of drug-DNA interactions: an in vitro transcription assay. Biochemistry, 25, 7355–7362.
Sobell,H.M. and Jain,S.C. (1972) Stereochemistry of actinomycin binding to DNA II. Detailed molecular model of actinomycin–DNA complex and its implication. J. Mol. Biol., 68, 21–34.
Kamitori,S. and Takusagawa,F. (1992) Crystal structure of the 2:1 complex between d(GAAGCTTC) and the anticancer drug actinomycin D. J. Mol. Biol., 225, 445–456.
Snyder,J.G., Hartman,N.G., D’Estantoit,B.L., Kennard,O., Remeta,D.P. and Breslauer,K.P. (1989) Binding of actinomycin D to DNA: evidence for a nonclassical high-affinity binding mode that does not require GpC sites. Proc. Natl Acad. Sci. USA, 86, 3968–3972.
Rill,R.L., Marsch,G.A. and Graves,D.E. (1989) 7-Azido-actinomycin D: a photoaffinity probe of the sequence specificity of DNA binding by actinomycin D. J. Biomol. Struct. Dyn., 7, 591–605.
Bailey,S.A., Graves,D.E. and Rill,R. (1994) Binding of actinomycin D to the T(G)nT motif of double-stranded DNA: determination of the guanine requirement in nonclassical, non-GpC binding sites. Biochemistry, 33, 11493–11500.
Wadkins,R.M. and Jovin,T.M. (1991) Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA. Biochemistry, 30, 9469–9478.
Hsieh,Y.L., Li,Y.T. and Henion,J.D. (1994) Studies of non-covalent interactions of actinomycin D with single-stranded oligodeoxynucleotides by ion spray mass spectrometry and tandem mass spectrometry. Biol. Mass Spectrom., 116, 272–276.
Wadkins,R.M., Jares-Erijman,E.A., Klement,R., Rudiger,A. and Jovin,T.M. (1996) Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and 1H NMR spectroscopy. J. Mol. Biol., 262, 53–68.
Rill,R.L. and Hecker,K.H. (1996) Sequence-specific actinomycin D binding to single-stranded DNA inhibits HIV reverse transcriptases and other polymerases. Biochemistry, 35, 3525–3533.
Wadkins,R.M., Vladu,B. and Tung,C.-S. (1998) Actinomycin D binds to metastable hairpins in single-stranded DNA. Biochemistry, 37, 11915–11923.
Yoo,H. and Rill,R.L. (2001) Actinomycin D binding to unstructured, single-stranded DNA. J. Mol. Recognit., 14, 145–150.
Chen,F.-M. and Sha,F. (2001) Actinomycin D binds strongly to d(TGTCATTG), a single-stranded DNA devoid of GpC sites. Biochemistry, 40, 5218–5225.
Chen,F.-M., Sha,F., Chin,K.-H. and Chou,S.-H. (2003) Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCTnG) and d(TGTnGTCT). Biophys. J., 84, 432–439.
Chou,S.-H., Chin,K.-H. and Chen,F.-M. (2002) Looped-out and perpendicular: deformation of Watson-Crick base pair associated with actinomycin D binding. Proc. Natl Acad. Sci. USA, 99, 6625–6630.
Chin,K.-H., Chen,F.M. and Chou,S.-H. (2003) Solution structure of actinomycin D/5'-CCGTTTTGTGG-3' complex: drug interaction with tandem G·T mismatches and hairpin loop backbone. Nucleic Acids Res., 31, 2622–2629.
Su,L. (1995) Conformations of single stranded DNA oligonucleotides and their actinomycin D complexes studies by high resolution 1H NMR spectroscopy. PhD dissertation, Florida State University, Tallahassee, FL.
Fasman,G.D. (ed.) (1975) CRC Handbook of Biochemistry and Molecular Biology, 3rd Edn. Chemical Rubber Publishing Co., Cleveland, OH, Vol. I, p. 589.
Chou,S.-H., Zhu,L., Gao,Z., Cheng,J.-W. and Reid,B.R. (1996) Hairpin loops consisting of single adenine residues closed by sheared A:A and G:G pairs formed by the DNA triplets AAA and GAG: solution structure of the d(GTACAAAGTAC) hairpin. J. Mol. Biol., 264, 981–1001.
Chou,S.-H., Tseng,Y.-Y. and Wang,S.-W. (1999) Stable sheared A:C pair in DNA hairpins. J. Mol. Biol., 287, 301–313.
Krugh,T.R., Mooberry,E.S. and Chiao,Y.-C.C. (1977) Proton magnetic resonance studies of actinomycin D complexes with mixtures of nucleotides as models for the binding of drug to DNA. Biochemistry, 16, 740–747.
Liu,X., Chen,H. and Patel,D.J. (1991) Solution structure of actinomycin–DNA complexes: drug intercalation at isolated G-C sites. J. Biomol. NMR, 1, 323–347.
Shuman,S. (1995) Vaccinia virus DNA ligase: specificity, fidelity and inhibition. Biochemistry, 34, 16138–16147.
Tuteja,N., Phan,T.N., Tuteja,R., Ochem,A. and Falaschi,A. (1997) Inhibition of DNA unwinding and ATPase activities of human DNA helicase II by chemotherapeutic agents. Biochem. Biophys. Res. Commun., 236, 636–640.
Davis,W.R., Gabbara,S., Hupe,D. and Peliska,J.A. (1998) Actinomycin D inhibition of DNA strand transfer reaction catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein. Biochemistry, 37, 14213–14221.
Guo,J., Wu,T., Bes,J., Henderson,L.E. and Levin,J.G. (1988) Actinomycin D inhibits human immunodeficiency virus type 1 minus-strand transfer in in vitro and endogenous reverse transcriptase assays. J. Virol., 72, 6716–6724.
Jeeninga,R.E., Huthoff,H.T., Gultyaev,A.P. and Berkhout,B. (1998) The mechanism of actinomycin D-mediated inhibition of HIV-1 reverse transcription. Nucleic Acids Res., 26, 5472–5479.
Gabbara,S., Davis,W.R., Hupe,L. and Peliska,J.A. (1999) Inhibitors of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Biochemistry, 38, 13070–13076.(Fu-Ming Chen*, Feng Sha, Ko-Hsin Chin1 a)