Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ～15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.     

Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA

Base excision DNA repair (BER) is fundamentally important in handling diverse lesions produced as a result of the intrinsic instability of DNA or by various endogenous and exogenous reactive species. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. BER funnels diverse base lesions into a common intermediate, apurinic/apyrimidinic (AP) sites. The repair of AP sites is initiated by the major human AP endonuclease, Ape1, or by AP lyase activities associated with some DNA glycosylases. Subsequent steps follow either of two distinct BER subpathways distinguished by repair DNA synthesis of either a single nucleotide (short-patch BER) or multiple nucleotides (long-patch BER). As the major repair mode for regular AP sites, the short-patch BER pathway removes the incised AP lesion, a 5'-deoxyribose-5-phosphate moiety, and replaces a single nucleotide using DNA polymerase (Polbeta). However, short-patch BER may have difficulty handling some types of lesions, as shown for the C1'-oxidized abasic residue, 2-deoxyribonolactone (dL). Recent work indicates that dL is processed efficiently by Ape1, but that short-patch BER is derailed by the formation of stable covalent crosslinks between Ape1-incised dL and Polbeta. The long-patch BER subpathway effectively removes dL and thereby prevents the formation of DNA-protein crosslinks. In coping with dL, the cellular choice of BER subpathway may either completely repair the lesion, or complicate the repair process by forming a protein-DNA crosslink.     

Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites in DNA by human cell extracts.

Base excision repair (BER) pathway is the major cellular process for removal of endogenous base lesions and apurinic/apyrimidinic (AP) sites in DNA. There are two base excision repair subpathways in mammalian cells, characterized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide" (one nucleotide incorporated) and the "long-patch" (several nucleotides incorporated) BER pathways. Proliferating cell nuclear antigen (PCNA) is known to be an essential factor in long-patch base excision repair. We have studied the role of replication protein A (RPA) in PCNA-dependent, long-patch BER of AP sites in human cell extracts. PCNA and RPA were separated from the other BER proteins by fractionation of human whole-cell extract on a phosphocellulose column. The protein fraction PC-FII (phosphocellulose fraction II), which does not contain RPA and PCNA but otherwise contains all core BER proteins required for PCNA-dependent BER (AP endonuclease, DNA polymerases delta, beta and DNA ligase, and FEN1 endonuclease), had reduced ability to repair plasmid DNA containing AP sites. Purified PCNA or RPA, when added separately, could only partially restore the PC-FII repair activity of AP sites. However, additions of both proteins together greatly stimulated AP site repair by PC-FII. These results demonstrate a role for RPA in PCNA-dependent BER of AP sites.     

Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.     

Influence of poly(ADP-ribose) polymerase-1 and its apoptotic 24-kD fragment on repair of DNA duplexes in bovine testis nuclear extract

Effects of exogenous proteins poly(ADP-ribose) polymerase-1 (PARP1) and its 24-kD proteolytic fragment (p24) on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. These damaged DNAs are repaired by the long-patch or short-patch subpathways of base excision repair (BER), respectively. Exogenous PARP1 and p24 decreased the efficiency of gap filling DNA synthesis for both duplexes, but did not influence the ligation stage in the repair of DNA duplex by the short-patch subpathway. Under the same conditions, these proteins inhibited strand-displacement DNA synthesis and decreased the efficiency of the flap endonuclease 1 (FEN1)-catalyzed endonuclease reaction in the nuclear extract, blocking repair of DNA duplex by the long-patch subpathway. Addition of exogenous PARP1 and p24 also reduced the efficiency of UV light crosslinking of extract BER proteins to the photoreactive BER intermediates carrying a nick. Thus, PARP1 and p24 interact with DNA intermediates of BER and compete with nuclear extract proteins for binding to DNA. The interaction of PARP1 and p24 with DNA intermediates of the long-patch subpathway of BER resulted in inhibition of subsequent stages of the repair mediated by this mechanism. However, on recovery of the intact structure of DNA duplex by the short-patch subpathway, PARP1 and p24 suppressed the repair of the one nucleotide gap less efficiently and failed to influence the final stage of the repair, ligation.     

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.     

Mitochondrial base excision repair of uracil and AP sites takes place by single-nucleotide insertion and long-patch DNA synthesis

Base excision repair (BER) corrects a variety of small base lesions in DNA. The UNG gene encodes both the nuclear (UNG2) and the mitochondrial (UNG1) forms of the human uracil-DNA glycosylase (UDG). We prepared mitochondrial extracts free of nuclear BER proteins from human cell lines. Using these extracts we show that UNG is the only detectable UDG in mitochondria, and mitochondrial BER (mtBER) of uracil and AP sites occur by both single-nucleotide insertion and long-patch repair DNA synthesis. Importantly, extracts of mitochondria carry out repair of modified AP sites which in nuclei occurs through long-patch BER. Such lesions may be rather prevalent in mitochondrial DNA because of its proximity to the electron transport chain, the primary site of production of reactive oxygen species. Furthermore, mitochondrial extracts remove 5' protruding flaps from DNA which can be formed during long-patch BER, by a "flap endonuclease like" activity, although flap endonuclease (FEN1) is not present in mitochondria. In conclusion, combined short- and long-patch BER activities enable mitochondria to repair a broader range of lesions in mtDNA than previously known.     

Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1

Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.     

Futile short-patch DNA base excision repair of adenine:8-oxoguanine mispair

8-Oxo-7, 8-dihydrodeoxyguanosine (8-oxo-dG), one of the representative oxidative DNA lesions, frequently mispairs with the incoming dAMP during mammalian DNA replication. Mispaired dA is removed by post-replicative base excision repair (BER) initiated by adenine DNA glycosylase, MYH, creating an apurinic (AP) site. The subsequent mechanism ensuring a dC:8-oxo-dG pair, a substrate for 8-oxoguanine DNA glycosylase (OGG1), remains to be elucidated. At the nucleotide insertion step, none of the mammalian DNA polymerases examined exclusively inserted dC opposite 8-oxo-dG that was located in a gap. AP endonuclease 1, which possesses 3′→5′ exonuclease activity and potentially serves as a proofreader, did not discriminate dA from dC that was located opposite 8-oxo-dG. However, human DNA ligases I and III joined 3′-dA terminus much more efficiently than 3′-dC terminus when paired to 8-oxo-dG. In reconstituted short-patch BER, repair products contained only dA opposite 8-oxo-dG. These results indicate that human DNA ligases discriminate dC from dA and that MYH-initiated short-patch BER is futile and hence this BER must proceed to long-patch repair, even if it is initiated as short-patch repair, through strand displacement synthesis from the ligation-resistant dC terminus to generate the OGG1 substrate, dC:8-oxo-dG pair.     

Exchangeability of mammalian DNA ligases between base excision repair pathways

In mammalian cells, DNA ligase IIIalpha and DNA ligase I participate in the short- and long-patch base excision repair pathways, respectively. Using an in vitro repair assay employing DNA ligase-depleted cell extracts and DNA substrates containing a single lesion repaired either through short-patch (regular abasic site) or long-patch (reduced abasic site) base excision repair pathways, we addressed the question whether DNA ligases are specific to each pathway or if they are exchangeable. We find that immunodepletion of DNA ligase I did not affect the short-patch repair pathway but blocked long-patch repair, suggesting that DNA ligase IIIalpha is not able to substitute DNA ligase I during long-patch repair. In contrast, immunodepletion of DNA ligase IIIalpha did not significantly affect either pathway. Moreover, repair of normal abasic sites in wild-type and X-ray cross-complementing gene 1 (XRCC1)-DNA ligase IIIalpha-immunodepleted cell extracts involved similar proportions of short- and long-patch repair events. This suggests that DNA ligase I was able to efficiently substitute the XRCC1-DNA ligase IIIalpha complex during short-patch 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.     

Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair

There exist two major base excision DNA repair (BER) pathways, namely single-nucleotide or “short-patch” (SP-BER), and “long-patch” BER (LP-BER). Both pathways appear to be involved in the repair of small base lesions such as uracil, abasic sites and oxidized bases. In addition to DNA polymerase β (Polβ) as the main BER enzyme for repair synthesis, there is evidence for a minor role for DNA polymerase lambda (Polλ) in BER. In this study we explore the potential contribution of Polλ to both SP- and LP-BER in cell-free extracts. We measured BER activity in extracts of mouse embryonic fibroblasts using substrates with either a single uracil or the chemically stable abasic site analog tetrahydrofuran residue. The addition of purified Polλ complemented the pronounced BER deficiency of POLB-null cell extracts as efficiently as did Polβ itself. We have developed a new approach for determining the relative contributions of SP- and LP-BER pathways, exploiting mass-labeled nucleotides to distinguish single- and multinucleotide repair patches. Using this method, we found that uracil repair in wild-type and in Polβ-deficient cell extracts supplemented with Polλ was ∼80% SP-BER. The results show that recombinant Polλ can contribute to both SP- and LP-BER. However, endogenous Polλ, which is present at a level ˜50% that of Polβ in mouse embryonic fibroblasts, appears to make little contribution to BER in extracts. Thus Polλ in cells appears to be under some constraint, perhaps sequestered in a complex with other proteins, or post-translationally modified in a way that limits its ability to participate effectively in BER.     

A quantitative model of human DNA base excision repair. I. Mechanistic insights

Base excision repair (BER) is a multistep process involving the sequential activity of several proteins that cope with spontaneous and environmentally induced mutagenic and cytotoxic DNA damage. Quantitative kinetic data on single proteins of BER have been used here to develop a mathematical model of the BER pathway. This model was then employed to evaluate mechanistic issues and to determine the sensitivity of pathway throughput to altered enzyme kinetics. Notably, the model predicts considerably less pathway throughput than observed in experimental in vitro assays. This finding, in combination with the effects of pathway cooperativity on model throughput, supports the hypothesis of cooperation during abasic site repair and between the apurinic/apyrimidinic (AP) endonuclease, Ape1, and the 8-oxoguanine DNA glycosylase, Ogg1. The quantitative model also predicts that for 8-oxoguanine and hydrolytic AP site damage, short-patch Polβ-mediated BER dominates, with minimal switching to the long-patch subpathway. Sensitivity analysis of the model indicates that the Polβ-catalyzed reactions have the most control over pathway throughput, although other BER reactions contribute to pathway efficiency as well. The studies within represent a first step in a developing effort to create a predictive model for BER cellular capacity.     

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.     

Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.     

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.     

The mechanism of base excision repair in Chlamydiophila pneumoniae

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.     

Long-patch base excision DNA repair of 2-deoxyribonolactone prevents the formation of DNA-protein cross-links with DNA polymerase beta

Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1'-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase beta (polbeta). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polbeta. DPC formation was much less under repair than non-repair conditions, with extracts of either polbeta-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5'-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polbeta and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of >or=2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.     

Interaction of human AP endonuclease 1 with flap endonuclease 1 and proliferating cell nuclear antigen involved in long-patch base excision repair

To understand the mechanism involved in the coordination of the sequential repair reactions that lead to long-patch BER, we have investigated interactions between proteins involved in this pathway. We find that human AP endonuclease 1 (APE1) physically interacts with flap endonuclease 1 (FEN1) and with proliferating cell nuclear antigen. An oligonucleotide substrate containing a reduced abasic site, which was pre-incised with APE1, was employed to reconstitute the excision step of long-patch BER with purified human DNA polymerase beta and FEN1. We demonstrate that addition of APE1 to the excision reaction mixture slightly (1.5-2-fold) stimulates the removal of the displaced flap by FEN1. These results suggest the possibility that long-patch BER is coordinated and directed by protein-protein interactions.