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J Bacteriol. 2011 August; 193(15): 3815–3821.
PMCID: PMC3147505

Role of High-Fidelity Escherichia coli DNA Polymerase I in Replication Bypass of a Deoxyadenosine DNA-Peptide Cross-Link [down-pointing small open triangle]

Abstract

Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (N6-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past N6-dA-linked peptides. Based on these results, we hypothesized that another member of that family, Escherichia coli polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific N6-dA dodecylpeptide cross-link were created and utilized for in vitro DNA replication assays using E. coli DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using E. coli strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific N6-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past N6-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.

INTRODUCTION

DNA-protein cross-links represent a class of DNA lesions that are formed as a consequence of endogenous metabolic processes and exposure to various chemical toxicants, such as acrolein (1, 18). Although the prokaryotic nucleotide excision repair machinery can function directly on a subset of DNA-protein cross-links, it has been demonstrated to act more efficiently on DNA-peptide cross-links, the proteolytic degradation products of DNA-protein cross-links (20, 22). However, little is known about the replication past DNA-peptide cross-link lesions. This lack of knowledge is an obstacle to understanding how cells tolerate these lesions.

Unrepaired DNA-peptide cross-links are hypothesized to be substrates for replication bypass by DNA polymerases. Previously, we demonstrated that when a single-stranded shuttle vector containing a major groove-linked DNA-peptide cross-link was replicated in African green monkey kidney cells (COS7 cells), this lesion was only marginally miscoding (19). Such data show that in a mammalian system, a subset of DNA-peptide cross-links can be bypassed in a relatively error-free manner. Germane to these observations, we recently demonstrated that polymerase ν (Pol ν), an A family human polymerase possessing in vitro translesion DNA synthesis (TLS) activity, can efficiently and accurately bypass an N6-dA dodecylpeptide cross-link lesion (Fig. 1) (31).

Fig. 1.
Structure of a γ-HO-PdA-derived peptide cross-link. The DNA-peptide cross-link consists of a dodecyl-(Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr) peptide attached via an acrolein moiety at the N-6 position of dA.

In Escherichia coli, Pol III carries out DNA synthesis at the replication fork, while specialized DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V, mainly conduct replication bypass of DNA lesions (10). Pol I, a member of the A family of DNA polymerases, is the most abundant DNA polymerase in E. coli in the absence of exogenous stressors and has many important cellular functions, including the processing of Okazaki fragments and DNA repair (25). In addition, Pol I has been shown to be capable of catalyzing replication bypass of various N6-dA-linked DNA lesions, ranging from a small styrene oxide-induced DNA adduct to more bulky benzo[a]pyrene-induced DNA adducts (5, 17).

To determine if high-fidelity bypass of an N6-dA dodecylpeptide cross-link by human Pol ν could be extrapolated to other A family polymerases, we evaluated the ability of E. coli Pol I and its exonuclease-deficient Klenow fragment (KFexo) to replicate past this lesion in vitro, and for comparative purposes, we conducted similar analyses with all other E. coli DNA polymerases. In addition, the efficiency and fidelity of intracellular TLS past the cross-link were assessed using either wild-type E. coli or an isogenic triple mutant that lacks DNA damage-inducible polymerases.

MATERIALS AND METHODS

Materials.

[γ-32P]ATP was purchased from PerkinElmer Life Sciences (Waltham, MA). P-6 Bio-Spin columns were obtained from Bio-Rad (Hercules, CA). T4 polynucleotide kinase, Pol I (10,000 U/ml), and KFexo (5,000 U/ml) were purchased from New England BioLabs (Beverly, MA). Sodium cyanoborohydride was obtained from Sigma (St. Louis, MO). Slide-A-Lyzer dialysis cassettes with a molecular weight cutoff of 10,000 were purchased from Thermo Scientific (Rockford, IL). The peptide Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr (KFHEKHHSHRGY) was obtained from Sigma-Genosys (St. Louis, MO). E. coli Pol II (4), Pol IV (14, 29), UmuD′2C (3), and RecA (6) were purified as previously described. The Pol III replicase (Pol III*), a holoenzyme that contains two molecules of Pol III core connected by the γ complex clamp loader but lacks the β subunit sliding clamp, was a generous gift from Michael O'Donnell (Rockefeller University, New York, NY) and was purified and reconstituted as previously described (21, 24, 26, 28). Pol κ was purchased from Enzymax, LLC (Lexington, KY).

Bacterial strains.

The Pol II-, IV-, and V-deficient strain SF2018 (ZK126 polB::Spcr dinB::Kanr umuDC::Camr) is derived from the E. coli K-12 strain ZK126 (W3110 ΔlacU169 tna-2) (33). This triple mutant strain was constructed by bacteriophage P1 transduction into ZK126, using the following donor strains: SF2003 (polB::Spcr), SF2006 (dinB::Kanr), and SF2009 (umuDC::Camr) (32). The original donor alleles and strains for the polymerase mutants mentioned above were as follows: for SF2003, SH2101 (polBΔ1::Ω Sm-Sp) (2); for SF2006, RW626 (dinB::Kan); and for SF2009, RW82 (umuDC::Cam) (both RW626 and RW82 were generous gifts from Roger Woodgate [National Institutes of Health, Bethesda, MD]). The triple mutant was selected by growing cells in the presence of spectinomycin (100 μg/ml), chloramphenicol (30 μg/ml), and kanamycin (50 μg/ml).

Oligodeoxynucleotide synthesis.

Nondamaged oligodeoxynucleotides were synthesized by the Molecular Microbiology and Immunology Research Core Facility at Oregon Health & Science University (Portland, OR). An oligodeoxynucleotide containing γ-HO-PdA (γ-hydroxypropanodeoxyadenosine) was a generous gift from Carmelo J. Rizzo (Vanderbilt University, Nashville, TN). An oligodeoxynucleotide containing an N6-dA dodecylpeptide cross-link (Fig. 1) (5′-GCTAGTACTCGTCGACAATTCCGTATCCAT-3′) at the underlined nucleotide was prepared according to a previously published procedure (31).

DNA replication assays.

For replication assays, primers were designed for conditions with either a running or standing start, where the primer 3′-OH was positioned three nucleotides upstream of the lesion site (−3 primer) or immediately prior to the lesion site (−1 primer), respectively. The sequences of the −3 and −1 primers were 5′-AAAATGGATACGGAAT-3′ and 5′-AAAATGGATACGGAATTG-3′, respectively. DNA replication assays with Pol III*, Pol I, KFexo, Pol II, Pol IV, and human Pol κ were carried out in reaction mixtures containing 25 mM Tris-HCl (pH 7.5), 10% (vol/vol) glycerol, 100 μg/ml bovine serum albumin, and 5 mM dithiothreitol. The Pol III*-, Pol I-, KFexo-, and Pol κ-catalyzed reactions were carried out in the presence of 8 mM MgCl2; the Pol II- and Pol IV-catalyzed reactions were carried out in the presence of 5 mM MgCl2. DNA replication assays with UmuD′2C were carried out in reaction mixtures containing 20 mM Tris-HCl (pH 7.5), 8 mM MgCl2, 4% (vol/vol) glycerol, 10 mM sodium glutamate, 5 mM dithiothreitol, 100 μM EDTA, 1 μM single-stranded 36-mer oligodeoxynucleotide, 6 μM RecA, and 0.5 mM adenosine 5′-[γ-thio]triphosphate. The single-stranded 36-mer oligodeoxynucleotide and RecA were preincubated in reaction buffer in the presence of adenosine 5′-[γ-thio]triphosphate at 37°C for 3 min. Reactions were initiated by the addition of deoxynucleoside triphosphates (dNTPs) and UmuD′2C. All reactions were carried out at 37°C. DNA substrate, DNA polymerase, and dNTP concentrations, as well as reaction times, are given in the figure legends. Reaction products were resolved through 15% acrylamide denaturing gels containing 8 M urea and were visualized using a PhosphorImager screen. The percentage of primer extension was determined by calculating the ratio of the amount of extended primers beyond the adducted or corresponding unadducted base to the total amount of primers (unextended primers plus extended primers up to and opposite the site of the adduct plus extended primers beyond the site of the adduct). Quantifications were done using ImageQuant 5.2 software.

Replication of DNAs containing site-specific N6-dA dodecylpeptide cross-link in E. coli.

The pMS2 shuttle vector was the generous gift of Masaaki Moriya (State University of New York, Stony Brook, NY). A single-stranded pMS2 vector containing a site-specific N6-dA dodecylpeptide cross-link (pMS2-XL12) was constructed following a previously published protocol (21). Initially, individual transformations with either unadducted reference pBR322 plasmid or pMS2-XL12 plasmid were carried out using a wild-type E. coli strain to determine the ratio of plasmid concentrations that would result in approximately equal numbers of transformants. The exact ratio of plasmids used for the transformations was not possible to calculate because following ligation of the lesion-containing insert into the pMS2 vector, the percentage of closed circular plasmids with the insert could not be determined accurately.

Following determination of a desirable ratio, the unadducted pBR322 and lesion-containing pMS2-XL12 plasmids were mixed and used to transform wild-type E. coli and an isogenic strain (SF2018 [Pol II Pol IV Pol V]). The transformants were selected overnight on LB agar plates containing ampicillin, and individual colonies were grown first in LB broth containing ampicillin (100 μg/ml) in 96-well plates at 37°C. After 6 h of incubation, a 20-μl aliquot from each well was transferred to other plates containing LB broth with tetracycline (12.5 μg/ml) and incubated overnight at 37°C. This procedure allowed the distinction of the tetracycline-sensitive pMS2 transformants from the tetracycline-resistant pBR322 transformants. In order to identify the population of transformants that originated from insert-containing pMS2-XL12 vectors, tetracycline-sensitive colonies were subjected to differential hybridization as previously described (21). As a probe, we used the oligodeoxynucleotide 5′-TACCAGCGATGCTAGTACT-3′, which hybridizes to the 5′ junction of the pMS2 backbone and the lesion-containing 30-mer oligodeoxynucleotide insert. To ensure the accuracy of this hybridization screen, plasmids from approximately 10% of colonies that did not hybridize with this probe were isolated and sequenced. The numbers of pBR322 and pMS2-XL12 transformants in the wild-type and Pol II Pol IV Pol V strains were determined, and the relative transformation efficiency of pMS2-XL12 in each strain was calculated by normalizing the transformation efficiency of pMS2-XL12 to that of pBR322. Mutational analyses were carried out by transforming the wild-type and Pol II Pol IV Pol V strains with a pMS2 vector containing either an unmodified or N6-dA dodecylpeptide cross-link-containing 30-mer oligonucleotide. The probe that was complementary to the DNA sequences encompassing the lesion, assuming no mutations were introduced, was used for differential hybridization. Vectors that did not hybridize to the probe were isolated by use of Qiagen plasmid minipreparation kits and digested with ScaI restriction endonuclease. Since both the original pMS2 vector and the insert had an ScaI recognition site, two DNA fragments were generated (~1.9 and ~3.2 kb) from vectors that contained inserted sequences. Subsequently, such vectors were analyzed by DNA sequencing.

RESULTS

Pol III*-catalyzed replication is blocked by N6-dA dodecylpeptide cross-link in vitro.

It has been shown previously that Saccharomyces cerevisiae replicative Pol δ cannot efficiently bypass an N6-dA dodecylpeptide cross-link (31). However, the ability of prokaryotic replicative polymerases to bypass this lesion has not been investigated. Therefore, primer extension reactions were conducted using nondamaged and N6-dA dodecylpeptide cross-link-containing templates with Pol III* (holoenzyme that lacks the β subunit sliding clamp). The results revealed that Pol III* could not bypass the cross-link because it was severely blocked at a site one nucleotide prior to the lesion, with only 2% of primers extended beyond the adducted site, compared to 65% with an unadducted template (Fig. 2).

Fig. 2.
Replication blockage of Pol III* by N6-dA dodecylpeptide cross-link. Primer extension reactions were catalyzed by 2 nM Pol III* under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked (XL12) substrates. ...

Replication bypass of N6-dA dodecylpeptide cross-link by Pol I and KFexo in vitro.

In order to test the hypothesis that Pol I could be involved in replication bypass of the N6-dA dodecylpeptide cross-link, primer extension reactions were carried out using Pol I. As shown in Fig. 3 A, Pol I fully extended the primers annealed to the adducted template; under the conditions used, minimal blockage of DNA synthesis was observed one nucleotide prior to the lesion. As evident from the bar graph, the percentages of primers that were extended beyond the adducted site and the corresponding unadducted site were comparable; specifically, 63% and 51% primer extension was observed with a low concentration of Pol I on unadducted and adducted templates, respectively. Thus, Pol I can catalyze replication bypass of a large N6-dA peptide cross-link.

Fig. 3.
Replication bypass of N6-dA dodecylpeptide cross-link by Pol I and KFexo. (A) Primer extension reactions were catalyzed by 0.32 mU or 1.6 mU Pol I under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide ...

In order to assess the identity of the nucleotide incorporated opposite the lesion, single-nucleotide incorporation reactions were conducted with KFexo. KFexo was used instead of Pol I in this experiment because the proofreading exonuclease activity of Pol I could potentially remove the misincorporated nucleotide, and thus the fidelity of nucleotide incorporation would be overestimated. In the presence of a 1 μM concentration of each dNTP individually, only the correct nucleotide, dT, was incorporated opposite the lesion (Fig. 3B), demonstrating that this cross-link can be bypassed accurately by KFexo and suggesting that Pol I-catalyzed replication past the lesion is error-free.

Replication bypass of N6-dA dodecylpeptide cross-link by DNA damage-inducible polymerases Pol II, Pol IV, and Pol V in vitro.

Since DNA damage-inducible polymerases are known to be involved in bypass of many lesions, the ability of these polymerases to bypass DNAs containing an N6-dA dodecylpeptide cross-link was examined. Primer extension reactions were carried out with Pol II, Pol IV, and Pol V (UmuD′2C and RecA complex). Pol IV could not efficiently bypass this lesion. It was strongly blocked at a site one nucleotide prior to the lesion, with only 11% of primers being extended beyond the adducted site, even at the highest concentration of Pol IV used. This is in contrast to 90% primer extension beyond the corresponding unadducted site (Fig. 4).

Fig. 4.
Replication blockage of Pol IV by N6-dA dodecylpeptide cross-link. Primer extension reactions were catalyzed by 1.25 nM and 5 nM Pol IV under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked (XL12) ...

Pol V also catalyzed inefficient bypass of the N6-dA dodecylpeptide cross-link. It could extend primers annealed to the adducted template up to the lesion, but the percentage of primers extended beyond the adducted site was only 4%, compared to 44% beyond the unadducted site (Fig. 5 A). However, this limited replication bypass appeared to be accurate, because only the correct nucleotide (dT) was incorporated opposite the lesion (Fig. 5B).

Fig. 5.
Replication bypass of N6-dA dodecylpeptide cross-link by Pol V. (A) Primer extension reactions were catalyzed by 200 nM UmuD′2C under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked (XL12) ...

Relative to Pol IV and Pol V, Pol II was significantly more competent in bypass of the N6-dA dodecylpeptide cross-link. In particular, using a low concentration of Pol II, the percentages of primers extended beyond the corresponding unadducted and adducted sites were 48% and 13%, respectively (Fig. 6 A). As shown in Fig. 6B, single-nucleotide incorporation reactions revealed that only the correct nucleotide (dT) was incorporated opposite the lesion. Collectively, these data suggest that Pol II could be another polymerase involved in replication bypass of the N6-dA dodecylpeptide cross-link.

Fig. 6.
Replication bypass of N6-dA dodecylpeptide cross-link by Pol II. (A) Primer extension reactions were catalyzed by 0.5 nM and 5 nM Pol II under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked (XL12) ...

Efficiency and accuracy of replication of plasmid DNAs containing N6-dA dodecylpeptide cross-link in vivo.

Although our in vitro results showed that Pol II was able to bypass the N6-dA dodecylpeptide cross-link lesion, Pol I manifested a greater ability. In contrast to Pol II, Pol I is known to be expressed constitutively. Therefore, DNA damage-inducible polymerases may not be required for the replication bypass of this lesion, and Pol I may play a major intracellular role in catalyzing replication bypass of such large peptide cross-links.

In order to test whether DNA damage-inducible polymerases are required for replication bypass of the N6-dA dodecylpeptide cross-link, 30-mer oligodeoxynucleotides containing this lesion were ligated into the single-stranded pMS2 vector. A mixture of lesion-containing pMS2-XL12 vector and pBR322 nondamaged reference vector was prepared and transformed into either wild-type or Pol II PolIV PolV E. coli. The transformants were then selected for ampicillin resistance and tested for tetracycline resistance to identify tetracycline-sensitive pMS2-containing clones. Furthermore, colonies were identified by differential hybridization using a probe that hybridizes to the 5′ junction of pMS2 DNA and the insert sequence. The colonies containing the progeny of either pBR322 or pMS2-XL12 were counted, and the transformation efficiency of pMS2-XL12 vector relative to that of pBR322 vector was calculated for each strain. No differences in the transformation efficiency of pMS2-XL12 vector were observed between the wild-type and Pol II PolIV PolV strains (Fig. 7). These data demonstrate that DNA damage-inducible polymerases, including Pol II, are dispensable for bypass of an N6-dA dodecylpeptide cross-link in vivo.

Fig. 7.
Ability of E. coli strains to replicate DNA containing N6-dA dodecylpeptide cross-link. A mixture of nondamaged pBR322 vector and site-specifically modified single-stranded pMS2 vector (pMS2-XL12) was transformed into an E. coli wild-type strain and a ...

In vitro, Pol I bypassed the N6-dA dodecylpeptide cross-link in an error-free manner. In order to test whether accurate bypass of this lesion also takes place within cells, mutational analyses were conducted by transforming pMS2-XL12 vector into the wild type or the Pol II PolIV PolV strain. After overnight selection in the presence of ampicillin, 95 and 96 colonies of wild-type and Pol II PolIV PolV cells, respectively, were analyzed for mutations as described in Materials and Methods. In parallel experiments, 89 and 96 individual clones of wild-type and Pol II PolIV PolV cells, respectively, were tested following replication of the corresponding nondamaged control vectors. The results revealed that no mutations were introduced opposite either nondamaged or damaged dA, demonstrating the accuracy of TLS past the N6-dA dodecylpeptide cross-link in vivo (data not shown).

Pol I is known to be essential for ColE1-type plasmid replication (8, 15). Since pMS2 possesses a ColE1 origin (9), a Pol I-defective strain could not be used for the plasmid-based approach described above to directly assess the role of Pol I in bypass of DNA lesions. However, given the strong inhibitory effect of the N6-dA dodecylpeptide cross-link on Pol III*-catalyzed DNA synthesis in vitro (Fig. 2) and the efficient replication of plasmids containing this lesion in the Pol II PolIV PolV strain (Fig. 7), it can be inferred that Pol I likely plays a key role in bypass of such large major groove peptide cross-links.

DISCUSSION

In the current investigation, we demonstrated that Pol I and Pol II could replicate DNAs containing an N6-dA dodecylpeptide cross-link in an error-free manner in vitro, while Pol III* and Pol IV were strongly inhibited by this type of lesion. Although Pol V could fully and accurately extend a primer on a damaged substrate, its efficiency was low. Since Pol I is expressed constitutively, we proposed that Pol I in cells can carry out TLS past an N6-dA dodecylpeptide cross-link and that the DNA damage-inducible polymerases are not required. Consistent with this idea, in vivo studies using wild-type and Pol II Pol IV Pol V E. coli strains revealed that DNA damage-inducible polymerases were dispensable.

Although these data suggested the essential role of Pol I in replication bypass of the N6-dA dodecylpeptide cross-link, an involvement of alternative DNA polymerases in this process cannot be ruled out completely. In particular, Pol III and possibly other DNA polymerases may manifest higher bypass efficiencies in cells than those observed in vitro due to stimulation of bypass by the β sliding clamp and additional accessory factors. It is also important to recognize that the division of labor between DNA polymerases in replication bypass is expected to be more complex in the context of chromosomal DNA, as opposed to a site-specific single lesion on plasmid DNA. Following chemical exposure, multiple structurally different DNA lesions are likely to be formed that may trigger the SOS response. The contribution of Pol II to TLS past N6-dA polypeptide cross-links could be quite prominent under such conditions, since its intracellular levels are significantly increased (10). Pol IV, the most abundant E. coli DNA polymerase in stressed cells (10), may also be involved in replication bypass of lesions, displacing more efficient but less abundant DNA polymerases from the primer termini. Such a phenomenon was recently observed during double-strand-break repair (11). In addition, the location of the lesions on the chromosome may also influence the choice of polymerase, similar to a site-specific polymerase switch during spontaneous mutagenesis (7).

The active site of Pol I is constrained, and Pol I has a stringent requirement for accommodating nucleobase pairs with the correct Watson-Crick geometry (13, 30). Therefore, it is intriguing that Pol I was capable of bypassing a very large N6-dA dodecylpeptide cross-link that not only blocked replicative polymerases (E. coli Pol III* and yeast Pol δ) but also blocked the low-fidelity polymerases specialized in lesion bypass (E. coli Pol IV and Pol V and human Pol κ) (Fig. 8). One possible explanation may be that this lesion has conformational flexibility, with the bulky dodecylpeptide pointed away from the active site of Pol I, and has no effect on the Watson-Crick geometry. The DNA-peptide structure might resemble that of Lys-Trp-Lys-Lys linked at N2-dG via the N-terminal Lys through an acrolein moiety. In the latter structure, the Trp indolyl group does not intercalate into the DNA, while the C-terminal Lys is exposed to solvent, interacting minimally with the DNA (12). However, a detailed analysis of the mechanism of action of Pol I in the bypass of the N6-dA dodecylpeptide cross-link awaits the co-crystal structure determination of Pol I in complex with this lesion.

Fig. 8.
Replication blockage of human Pol κ by N6-dA dodecylpeptide cross-link. Primer extension reactions were catalyzed by 1 nM Pol κ under running-start conditions (−3 primer), using nondamaged (ND) and N6-dA dodecylpeptide cross-linked ...

With regard to the biological significance of N6-dA peptide cross-links, a previous report focused exclusively on the identification of E. coli DNA polymerases that can catalyze replication bypass of N2-dG peptide cross-links (21). The polymerases responsible for the replication bypass of N6-dA peptide cross-links, however, were not studied. The N6-dA peptide lesion used in this study was derived from the ring-opened aldehydic form of the acrolein-induced γ-HO-PdA adduct. Nuclear magnetic resonance spectroscopy analyses showed that γ-HO-PdA in the ring-opened form can be detected readily (23). This suggests that γ-HO-PdA could potentially interact with peptides, resulting in the formation of DNA-peptide cross-links. Such a mechanism would be conceptually similar to that for the aldehydic group of ring-opened γ-HO-PdG, which reacts with peptides and yields chemically identical lesions, except that the lesions are located in the minor groove of DNA (16). Consistent with this prediction, it was demonstrated that the peptide Lys-Trp-Lys-Lys is trapped efficiently and stably with oligodeoxynucleotides containing γ-HO-PdA in the presence of a reducing agent (19). Thus, although N6-dA peptide cross-links have not been identified in vivo to date, their biological relevance cannot be disregarded, and therefore the present investigation examining the identity of polymerases involved in the processing of these lesions has importance.

It is worth noting that in E. coli, TLS past N2-dG peptide cross-links requires the DNA damage-inducible polymerase Pol IV (21). In contrast, such specialized polymerases were not essential for replication bypass of the N6-dA dodecylpeptide cross-link. This is interesting given the fact that the previously investigated N2-dG dodecylpeptide cross-link is chemically identical to the N6-dA dodecylpeptide cross-link used here, except that the former lesion is positioned in the minor, not the major, groove of DNA. Germane to this observation, DNA polymerases of the A family interact with DNA at the minor groove (27). Thus, Pol I may have evolved to be particularly proficient in the bypass of major groove lesions, including relatively small adducts (5, 17) and large degradation products of DNA-protein cross-links.

In summary, we have shown the ability of E. coli Pol I to accurately replicate past an N6-dA dodecylpeptide cross-link in vitro and have generated data suggesting its role in bypass of this lesion in vivo. Since no data about the identity of E. coli DNA polymerases responsible for processing the major groove cross-links are available to date, the findings reported here promote our understanding of how cells maintain genome integrity upon induction of DNA-peptide and DNA-protein cross-links.

ACKNOWLEDGMENTS

We thank Carmelo J. Rizzo (Vanderbilt University, Nashville, TN) for the gift of γ-HO-PdA-containing oligodeoxynucleotides, Masaaki Moriya (State University of New York, Stony Brook, NY) for the gift of the pMS2 vector, Michael O'Donnell (Rockefeller University, New York, NY) for the gift of Pol III*, and Roger Woodgate (National Institutes of Health, Bethesda, MD) for the gifts of E. coli strains RW626 and RW82.

This work was supported by Public Health Service grants ES05355 (R.S.L.) and ES012259 (M.F.G.) from the National Institute of Environmental Health Sciences, grant CA106858 (R.S.L.) from the National Cancer Institute, grant GM21422 (M.F.G.) from the National Institute of General Medicine, and an NSF Career Award (MCB0237975).

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 May 2011.

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