Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.     

DNA joint dependence of pol X family polymerase action in nonhomologous end joining

DNA double strand breaks (DSBs) can be rejoined directly by the nonhomologous end-joining (NHEJ) pathway of repair. Nucleases and polymerases are required to promote accurate NHEJ when the terminal bases of the DSB are damaged. The same enzymes also participate in imprecise rejoining and joining of incompatible ends, important mutagenic events. Previous work has shown that the Pol X family polymerase Pol4 is required for some but not all NHEJ events that require gap filling in Saccharomyces cerevisiae. Here, we systematically analyzed DSB end configurations and found that gaps on both strands and overhang polarity are the principal factors that determine whether a joint requires Pol4. DSBs with 3'-overhangs and a gap on each strand strongly depended on Pol4 for repair, DSBs with 5'-overhangs of the same sequence did not. Pol4 was not required when 3'-overhangs contained a gap on only one strand, however. Pol4 was equally required at 3'-overhangs of all lengths within the NHEJ-dependent range but was dispensable outside of this range, indicating that Pol4 is specific to NHEJ. Loss of Pol4 did not affect the rejoining of DSBs that utilized a recessed microhomology or DSBs bearing 5'-hydroxyls but no gap. Finally, mammalian Pol X polymerases were able to differentially complement a pol4 mutation depending on the joint structure, demonstrating that these polymerases can participate in yeast NHEJ but with distinct properties.     

Pol3 is involved in nonhomologous end-joining in Saccharomyces cerevisiae

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.     

Mismatch tolerance by DNA polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere-telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3'-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand.     

Proofreading Activity of DNA Polymerase Pol2 Mediates 3′-End Processing during Nonhomologous End Joining in Yeast

Genotoxic agents that cause double-strand breaks (DSBs) often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ), but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3′ DNA ends, Pol2 is important for the recession of 3′ flaps that can form during imprecise pairing. Indeed, a mutation in the 3′-5′ exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3′ flaps. Thus, Pol2 performs a key 3′ end-processing step in NHEJ.     

Efficient processing of DNA ends during yeast nonhomologous end joining. Evidence for a DNA polymerase beta (Pol4)-dependent pathway.

Repair of DNA double strand breaks by nonhomologous end joining (NHEJ) requires enzymatic processing beyond simple ligation when the terminal bases are damaged or not fully compatible. We transformed yeast with a series of linearized plasmids to examine the role of Pol4 (Pol IV, DNA polymerase beta) in repair at a variety of end configurations. Mutation of POL4 did not impair DNA polymerase-independent religation of fully compatible ends and led to at most a 2-fold reduction in the frequency of joins that require only DNA polymerization. In contrast, the frequency of joins that also required removal of a 5'- or 3'-terminal mismatch was markedly reduced in pol4 (but not rev3, exo1, apn1, or rad1) yeast. In a chromosomal double strand break assay, pol4 mutation conferred a marked increase in sensitivity to HO endonuclease in a rad52 background, due primarily to loss of an NHEJ event that anneals with a 3'-terminal mismatch. The NHEJ activity of Pol4 was dependent on its nucleotidyl transferase function, as well as its unique amino terminus. Paradoxically, in vitro analyses with oligonucleotide substrates demonstrated that although Pol4 fills gaps with displacement of mismatched but not matched 5' termini, it lacks both 5'- and 3'-terminal nuclease activities. Pol4 is thus specifically recruited to perform gap-filling in an NHEJ pathway that must also involve as yet unidentified nucleases.     

Interaction of replication protein A and flap endonuclease 1 with DNA duplexes containing a nick or flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase beta (Pol beta) interaction with DNA. Using Pol beta, a photoreactive dTTP analog was added to the 3' end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5' end of a nick or a flap. Photoaffinity labeling of Pol beta substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol beta-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol beta during long-patch base excision repair.     

Strand displacement synthesis by yeast DNA polymerase ε

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.     

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.     

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site.To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.     

Efficiency and fidelity of human DNA polymerases λ and β during gap-filling DNA synthesis

The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.     

Multiple end joining mechanisms repair a chromosomal DNA break in fission yeast

Non-homologous end joining (NHEJ) is an important mechanism for repairing DNA double-strand breaks (DSBs). The fission yeast Schizosaccharomyces pombe has a conserved set of NHEJ factors including Ku, DNA ligase IV, Xlf1, and Pol4. Their roles in chromosomal DSB repair have not been directly characterized before. Here we used HO endonuclease to create a specific chromosomal DSB in fission yeast and examined the imprecise end joining events allowing cells to survive the continuous expression of HO. Our analysis showed that cell survival was significantly reduced in mutants defective for Ku, ligase IV, or Xlf1. Using Sanger sequencing and Illumina sequencing, we have characterized in depth the repair junction sequences in HO survivors. In wild type cells the majority of repair events were one-nucleotide insertions dependent on Ku, ligase IV, and Pol4. Our data suggest that fission yeast Pol4 is important for gap filling during NHEJ repair and can extend primers in the absence of terminal base pairing with the templates. In Ku and ligase IV mutants, the survivors mainly resulted from two types of alternative end joining events: one used microhomology flanking the HO site to delete sequences of hundreds to thousands of base pairs, the other rejoined the break using the HO-generated overhangs but also introduced one- or two-nucleotide base substitutions. The chromosomal repair assay we describe here should provide a useful tool for further exploration of the end joining repair mechanisms in fission yeast.     

Saccharomyces cerevisiae Sae2- and Tel1-dependent single-strand DNA formation at DNA break promotes microhomology-mediated end joining

Microhomology-mediated end joining (MMEJ) joins DNA ends via short stretches [5-20 nucleotides (nt)] of direct repeat sequences, yielding deletions of intervening sequences. Non-homologous end joining (NHEJ) and single-strand annealing (SSA) are other error prone processes that anneal single-stranded DNA (ssDNA) via a few bases (<5 nt) or extensive direct repeat homologies (>20 nt). Although the genetic components involved in MMEJ are largely unknown, those in NHEJ and SSA are characterized in some detail. Here, we surveyed the role of NHEJ or SSA factors in joining of double-strand breaks (DSBs) with no complementary DNA ends that rely primarily on MMEJ repair. We found that MMEJ requires the nuclease activity of Mre11/Rad50/Xrs2, 3' flap removal by Rad1/Rad10, Nej1, and DNA synthesis by multiple polymerases including Pol4, Rad30, Rev3, and Pol32. The mismatch repair proteins, Rad52 group genes, and Rad27 are dispensable for MMEJ. Sae2 and Tel1 promote MMEJ but inhibit NHEJ, likely by regulating Mre11-dependent ssDNA accumulation at DNA break. Our data support the role of Sae2 and Tel1 in MMEJ and genome integrity.     

DNA polymerase beta is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts.

The repair of oxidative base lesions in DNA is a coordinated chain of reactions that includes removal of the damaged base, incision of the phosphodiester backbone at the abasic sugar residue, incorporation of an undamaged nucleotide and sealing of the DNA strand break. Although removal of a damaged base in mammalian cells is initiated primarily by a damage-specific DNA glycosylase, several lyases and DNA polymerases may contribute to the later stages of repair. DNA polymerase beta (Pol beta) was implicated recently as the major polymerase involved in repair of oxidative base lesions; however, the identity of the lyase participating in the repair of oxidative lesions is unclear. We studied the mechanism by which mammalian cell extracts process DNA substrates containing a single 8-oxoguanine or 5,6-dihydrouracil at a defined position. We find that, when repair synthesis proceeds through a Pol beta-dependent single nucleotide replacement mechanism, the 5'-deoxyribosephosphate lyase activity of Pol beta is essential for repair of both lesions.     

Contributions of an endonuclease IV homologue to DNA repair in the African swine fever virus

We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3' mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3'-phosphatase and 3'-phosphodiesterase activities (in the context of a single-nucleotide gap), 3' --> 5' exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3' --> 5' exonuclease activity is shown to be highly dependent upon the identity of the nascent 3' base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5' moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.     

Role of the yeast DNA repair protein Nej1 in end processing during the repair of DNA double strand breaks by non-homologous end joining

DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.     

Promiscuous mismatch extension by human DNA polymerase lambda

DNA polymerase lambda (Pol λ) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol λ to extend mismatched primer-termini, either on ‘open’ template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5′-phosphate. Interestingly, Pol λ extended mismatches with an average efficiency of ≈10⁻² relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were ≈260-fold higher than on template-primer molecules. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer-terminus (2.40 Å) suggests that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol λ suggests a potential role as a ‘mismatch extender’ during non-homologous end joining (NHEJ), and possibly during translesion synthesis.     

Exonuclease of human DNA polymerase gamma disengages its strand displacement function

Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo^? (3′–5′ exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo^?, is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB.     

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair.     

Repair of abasic sites in DNA

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase beta adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase delta/epsilon and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase delta/epsilon is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.