Evaluating the effects of enhanced processivity and metal ions on translesion DNA replication catalyzed by the bacteriophage T4 DNA polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-1)). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn(2+) for Mg(2+) did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45.     

Human DNA polymerase beta mutations allowing efficient abasic site bypass

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.     

Repair of clustered DNA lesions. Sequence-specific inhibition of long-patch base excision repair be 8-oxoguanine

Ionizing radiation induces clustered DNA damage where two or more lesions are located proximal to each other on the same or opposite DNA strands. It has been suggested that individual lesions within a cluster are removed sequentially and that the presence of a vicinal lesion(s) may affect the rate and fidelity of DNA repair. In this study, we addressed the question of how 8-oxoguanine located opposite to normal or reduced abasic sites would affect the repair of these sites by the base excision repair system. We have found that an 8-oxoguanine located opposite to an abasic site does not affect either the efficiency or fidelity of repair synthesis by DNA polymerase beta. In contrast, an 8-oxoguanine located one nucleotide 3'-downstream of the abasic site significantly reduces both strand displacement synthesis supported by DNA polymerase beta or delta and cleavage by flap endonuclease of the generated flap, thus inhibiting the long-patch base excision repair pathway.     

DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase

Abasic (apurinic/apyrimidinic) sites are among the most abundant DNA lesions in humans, and they present a strong block to replication. They are also highly mutagenic because when replicative DNA polymerases manage to insert a nucleotide opposite the lesion, they prefer to insert an A. Rev1, a member of Y-family DNA polymerases, does not obey the A-rule. This enzyme inserts a C opposite an abasic lesion with much greater catalytic efficiency than an A, G, or T. We present here the structure of yeast Rev1 in ternary complex with DNA containing an abasic lesion and with dCTP as the incoming nucleotide. The structure reveals a mechanism of synthesis across an abasic lesion that differs from that in other polymerases. The lesion is driven to an extrahelical position, and the incorporation of a C is mediated by an arginine (Arg324) that is conserved in all known orthologs of Rev1, including humans. The hydrophobic cavity that normally accommodates the unmodified G is instead filled with water molecules. Since Gs are especially prone to depurination through a spontaneous hydrolysis of the glycosidic bond, the ability of Rev1 to stabilize an abasic lesion in its active site and employ a surrogate arginine to incorporate a C provides a unique means for the “error-free” bypass of this noninstructional lesion.     

Amino acid templating mechanisms in selection of nucleotides opposite abasic sites by a family a DNA polymerase

Cleavage of the N-glycosidic bond that connects the nucleobase to the backbone in DNA leads to abasic sites, the most frequent lesion under physiological conditions. Several DNA polymerases preferentially incorporate an A opposite this lesion, a phenomenon termed "A-rule." Accordingly, KlenTaq, the large fragment of Thermus aquaticus DNA polymerase I, incorporates a nucleotide opposite an abasic site with efficiencies of A > G > T > C. Here we provide structural insights into constraints of the active site during nucleotide selection opposite an abasic site. It appears that these confines govern the nucleotide selection mainly by interaction of the incoming nucleotide with Tyr-671. Depending on the nucleobase, the nucleotides are differently positioned opposite Tyr-671 resulting in different alignments of the functional groups that are required for bond formation. The distances between the α-phosphate and the 3'-primer terminus increases in the order A < G < T, which follows the order of incorporation efficiency. Additionally, a binary KlenTaq structure bound to DNA containing an abasic site indicates that binding of the nucleotide triggers a remarkable rearrangement of enzyme and DNA template. The ability to resolve the stacking arrangement might be dependent on the intrinsic properties of the respective nucleotide contributing to nucleotide selection. Furthermore, we studied the incorporation of a non-natural nucleotide opposite an abasic site. The nucleotide was often used in studying stacking effects in DNA polymerization. Here, no interaction with Tyr-761 as found for the natural nucleotides is observed, indicating a different reaction path for this non-natural nucleotide.     

Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2′-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis.     

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

DNA damage blocks DNA polymerase progression and increases miscoding. In this study, we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-Oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the mean modified efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.     

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.     

NGS-based analysis of base-substitution signatures created by yeast DNA polymerase eta and zeta on undamaged and abasic DNA templates in vitro

Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of base-substitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct base-substitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.     

Inactivation of the 3'-5' exonuclease of the replicative T4 DNA polymerase allows translesion DNA synthesis at an abasic site

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged "minicircle" DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.     

Snapshots of replication through an abasic lesion; structural basis for base substitutions and frameshifts

Dpo4 from S. Solfataricus, a DinB-like Y family polymerase, efficiently replicates DNA past an abasic lesion. We have determined crystal structures of Dpo4 complexed with five different abasic site-containing DNA substrates and find that translesion synthesis is template directed with the abasic site looped out and the incoming nucleotide is opposite the base 5' to the lesion. The ensuing DNA synthesis generates a -1 frameshift when the abasic site remains extrahelical. Template realignment during primer extension is also observed, resulting in base substitutions or even +1 frameshifts. In the case of a +1 frameshift, the extra nucleotide is accommodated in the solvent-exposed minor groove. In addition, the structure of an unproductive Dpo4 ternary complex suggests that the flexible little finger domain facilitates DNA orientation and translocation during translesion synthesis.     

Insights into the DNA repair process by the formamidopyrimidine-DNA glycosylase investigated by molecular dynamics

Formamidopyrimidine-DNA glycosylase (Fpg) identifies and removes 8-oxoguanine from DNA. All of the X-ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C-terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X-ray structure of Lactococcus lactis Fpg bound to an abasic site analog-containing DNA and of Bacillus stearothermophilus Fpg bound to 8-oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8-oxoG in its binding site. In addition, from our results, the conformation of the 8-oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X-ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.     

Electrostatic potential maps of damaged DNA studied by image analysis tools. 8-Oxoguanine and abasic site lesions

Changes of electrostatic potential around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. The electrostatic potential around the DNA fragments containing either the intact guanine-cytosine pair or 8-oxoguanine-cytosine or the guanine-abasic site was projected on a cylindrical surface around the double helix. The 2D maps of EP of intact and damaged DNA fragments were compared using image analysis methods. Occurrence of abasic site and 8-oxoguanine lesions were found to be reflected in the EP maps. In the case of the 8-oxoguanine lesion, the two phosphate groups and countercations of the damaged strand are moved away from the lesion in opposite directions, whereas they are moved in the same direction in the case of the abasic site lesion. The characteristic features of 8-oxoguanine lesion might be identified in the major groove, whereas the features of abasic site lesion the minor groove. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions

The A-rule in mutagenesis was originally proposed to explain the preponderance of X-->T mutations observed for abasic sites and UV damaged sites. It was deduced that when a polymerase was faced with a non-instructional lesion, typified by an abasic site, it would preferentially incorporate an A. In the absence of any other compelling explanation, any lesion causing an X-->T mutation has often been classified as non-instructional to account for its apparent lack of instructional ability. The A-rule and the classification of lesions as non-instructional were formulated before the active sites of any polymerases or the mechanism by which they synthesized DNA were known. Since then, much structural and kinetic data on DNA polymerases has emerged to suggest mechanistic explanations for the A-rule and the instructive and non-instructive behavior of lesions such as cis-syn dimers. Polymerases involved in the replication of undamaged DNA have highly constrained active sites that evolved to only accommodate the templating base and the complementary nucleotide and as a result are relatively intolerant of modifications that alter the size and shape of the nascent base pair. On the other hand, DNA damage bypass polymerases have much more open and less constrained active sites, which are much more tolerant of modifications. An otherwise instructional lesion would become non-instructional if it were unable to fit into the active site, and thereby behave transiently like an abasic site, leading to the insertion of whichever nucleotide is favored by the polymerase, generally an A. In this review, what is known about the active sites and mechanisms of replicative and DNA damage bypass polymerases will be discussed with regard to the A-rule and non-instructive behavior of lesions, typified by dipyrimidine photoproducts.     

DNA polymerase theta purified from human cells is a high-fidelity enzyme

With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3'-->5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta.     

Multiple solutions to inefficient lesion bypass by T7 DNA polymerase

We hypothesize that enzymatic switching during translesion synthesis (TLS) to relieve stalled replication forks occurs during transitions from preferential to disfavored use of damaged primer-templates, and that the polymerase or 3'-exonuclease used for each successive nucleotide incorporated is the one whose properties result in the highest efficiency and the highest fidelity of bypass. Testing this hypothesis requires quantitative determination of the relative lesion bypass ability of both TLS polymerases and major replicative polymerases. As a model of the latter, here we measure the efficiency and fidelity of cis-syn TT dimer and abasic site bypass using the structurally well-characterized T7 DNA polymerase. No bypass of either lesion occurred during a single round of synthesis, and the exonuclease activity of wild-type T7 DNA polymerase was critical in preventing TLS. When repetitive cycling of the exonuclease-deficient enzyme was allowed, limited bypass did occur but hundreds to thousands of cycles were required to achieve even a single bypass event. Analysis of TLS fidelity indicated that these rare bypass events involved rearrangements of the template and primer strands, insertions opposite the lesion, and combinations of these events, with the choice among these strongly depending on the sequence context of the lesion. Moreover, the presence of a lesion affected the fidelity of copying adjacent undamaged template bases, even when lesion bypass itself was correct. The results also indicate that a TT dimer presents a different type of block to the polymerase than an abasic site, even though both lesions are extremely potent blocks to processive synthesis. The approaches used here to quantify the efficiency and fidelity of TLS can be applied to other polymerase-lesion combinations, to provide guidance as to which of many possible polymerases is most likely to bypass various lesions in biological contexts.     

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.     

Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase

Accurate DNA replication involves polymerases with high nucleotide selectivity and proofreading activity. We show here why both fidelity mechanisms fail when normally accurate T7 DNA polymerase bypasses the common oxidative lesion 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8oG). The crystal structure of the polymerase with 8oG templating dC insertion shows that the O8 oxygen is tolerated by strong kinking of the DNA template. A model of a corresponding structure with dATP predicts steric and electrostatic clashes that would reduce but not eliminate insertion of dA. The structure of a postinsertional complex shows 8oG(syn).dA (anti) in a Hoogsteen-like base pair at the 3' terminus, and polymerase interactions with the minor groove surface of the mismatch that mimic those with undamaged, matched base pairs. This explains why translesion synthesis is permitted without proofreading of an 8oG.dA mismatch, thus providing insight into the high mutagenic potential of 8oG.     

Long patch base excision repair compensates for DNA polymerase β inactivation by the C4'-oxidized abasic site

The C4'-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents, has significant consequences for DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ~12-fold less efficiently than an apurinic/apyrimidinic lesion. DNA polymerase β induces the β-elimination of incised C4-AP in ternary complexes, duplexes, and single-stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after approximately seven turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5'-(2-phosphoryl-1,4-dioxobutane), which very efficiently irreversibly inhibits DNA polymerase β, the lesion is readily removed by strand displacement synthesis conducted by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion.