Domain specific interaction in the XRCC1-DNA polymerase beta complex

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase β (β-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain–domain interaction in the XRCC1-β-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I–linker–BRCT-II C-terminal fragment and the linker–BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact β-Pol and the β-Pol 31 kDa domain. The XRCC1-NTD_1–183 (residues 1–183) was found to bind β-Pol, the β-Pol 31 kDa domain and the β-Pol C-terminal palm-thumb (residues 140–335), and the interaction was further localized to XRCC1-NTD_1–157 (residues 1–157). The XRCC1-NTD_1–183-β-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36–355 and residues 1–159, were found to interact with β-Pol, the β-Pol 31 kDa domain, and the β-Pol C-terminal thumb-only domain polypeptides expressed from the respective β-Pol constructs. Neither the XRCC1-NTD_1–159, nor the XRCC1_36–355 polypeptide was found to interact with a β-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75–212) showed no interaction with β-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD_1–183 was found to bind β-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K_d of 0.4–2.4 µM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD_1–159 and β-Pol that is of an affinity comparable to other binding interactions involving β-Pol.     

Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1

The DNA damage dependence of poly(ADP-ribose) polymerase-2 (PARP-2) activity is suggestive of its implication in genome surveillance and protection. Here we show that the PARP-2 gene, mainly expressed in actively dividing tissues follows, but to a smaller extent, that of PARP-1 during mouse development. We found that PARP-2 and PARP-1 homo- and heterodimerize; the interacting interfaces, sites of reciprocal modification, have been mapped. PARP-2 was also found to interact with three other proteins involved in the base excision repair pathway: x-ray cross complementing factor 1 (XRCC1), DNA polymerase beta, and DNA ligase III, already known as partners of PARP-1. XRCC1 negatively regulates PARP-2 activity, as it does for PARP-1, while being a polymer acceptor for both PARP-1 and PARP-2. To gain insight into the physiological role of PARP-2 in response to genotoxic stress, we developed by gene disruption mice deficient in PARP-2. Following treatment by the alkylating agent N-nitroso-N-methylurea (MNU), PARP-2-deficient cells displayed an important delay in DNA strand breaks resealing, similar to that observed in PARP-1 deficient cells, thus confirming that PARP-2 is also an active player in base excision repair despite its low capacity to synthesize ADP-ribose polymers.     

XRCC1 interactions with base excision repair DNA intermediates

Abasic (AP) sites in DNA arise either spontaneously, or through glycosylase-catalyzed excision of damaged bases. Their removal by the base excision repair (BER) pathway avoids their mutagenic and cytotoxic consequences. XRCC1 coordinates and facilitates single-strand break (SSB) repair and BER in mammalian cells. We report that XRCC1, through its NTD and BRCT1 domains, has affinity for several DNA intermediates in BER. As shown by its capacity to form a covalent complex via Schiff base, XRCC1 binds AP sites. APE1 suppresses binding of XRCC1 to unincised AP sites however, affinity was higher when the DNA carried an AP-lyase- or APE1-incised AP site. The AP site binding capacity of XRCC1 is enhanced by the presence of strand interruptions in the opposite strand. Binding of XRCC1 to BER DNA intermediates could play an important role to warrant the accurate repair of damaged bases, AP sites or SSBs, in particular in the context of clustered DNA damage.     

XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair

XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIα (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.     

Imbalanced base excision repair in response to folate deficiency is accelerated by polymerase beta haploinsufficiency

The mechanism by which folate deficiency influences carcinogenesis is not well established, but a phenotype of DNA strand breaks, mutations, and chromosomal instability suggests an inability to repair DNA damage. To elucidate the mechanism by which folate deficiency influences carcinogenicity, we have analyzed the effect of folate deficiency on base excision repair (BER), the pathway responsible for repairing uracil in DNA. We observe an up-regulation in initiation of BER in liver of the folate-deficient mice, as evidenced by an increase in uracil DNA glycosylase protein (30%, p < 0.01) and activity (31%, p < 0.05). However, no up-regulation in either BER or its rate-determining enzyme, DNA polymerase beta (beta-pol) is observed in response to folate deficiency. Accordingly, an accumulation of repair intermediates in the form of DNA single strand breaks (37% increase, p < 0.03) is observed. These data indicate that folate deficiency alters the balance and coordination of BER by stimulating initiation without subsequently stimulating the completion of repair, resulting in a functional BER deficiency. In directly establishing that the inability to induce beta-pol and mount a BER response when folate is deficient is causative in the accumulation of toxic repair intermediates, beta-pol-haploinsufficient mice subjected to folate deficiency displayed additional increases in DNA single strand breaks (52% increase, p < 0.05) as well as accumulation in aldehydic DNA lesions (38% increase, p < 0.01). Since young beta-polhaploinsufficient mice do not spontaneously exhibit increased levels of these repair intermediates, these data demonstrate that folate deficiency and beta-pol haploinsufficiency interact to increase the accumulation of DNA damage. In addition to establishing a direct role for beta-pol in the phenotype expressed by folate deficiency, these data are also consistent with the concept that repair of uracil and abasic sites is more efficient than repair of oxidized bases.     

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair

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XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.     

XRCC1 and DNA polymerase beta interaction contributes to cellular alkylating-agent resistance and single-strand break repair

X-ray cross complementing 1 (XRCC1) protein has been suggested to bind to DNA single-strand breaks (SSBs) and organize protein interactions that facilitate efficient DNA repair. Using four site-specifically modified human XRCC1 mutant expression systems and functional complementation assays in Chinese hamster ovary (CHO) XRCC1-deficient EM9 cells, we evaluated the cellular contributions of XRCC1s proposed N-terminal domain (NTD) DNA binding and DNA polymerase beta (POLbeta) interaction activities. Results within demonstrate that the interaction with POLbeta is biologically important for alkylating agent resistance and SSB repair, whereas the proposed DNA binding function is not critical to these phenotypes. Our data favor a model where the interaction of XRCC1 with POLbeta contributes to efficient DNA repair in vivo, whereas its interactions with target DNA is biologically less relevant.     

FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair

In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.     

NuA4 acetyltransferase is required for efficient nucleotide excision repair in yeast

The nucleotide excision repair (NER) pathway is critical for removing damage induced by ultraviolet (UV) light and other helix-distorting lesions from cellular DNA. While efficient NER is critical to avoid cell death and mutagenesis, NER activity is inhibited in chromatin due to the association of lesion-containing DNA with histone proteins. Histone acetylation has emerged as an important mechanism for facilitating NER in chromatin, particularly acetylation catalyzed by the Spt-Ada-Gcn5 acetyltransferase (SAGA); however, it is not known if other histone acetyltransferases (HATs) promote NER activity in chromatin. Here, we report that the essential Nucleosome Acetyltransferase of histone H4 (NuA4) complex is required for efficient NER in Saccharomyces cerevisiae. Deletion of the non-essential Yng2 subunit of the NuA4 complex causes a general defect in repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast; in contrast, deletion of the Sas3 catalytic subunit of the NuA3 complex does not affect repair. Rapid depletion of the essential NuA4 catalytic subunit Esa1 using the anchor-away method also causes a defect in NER, particularly at the heterochromatic HML locus. We show that disrupting the Sds3 subunit of the Rpd3L histone deacetylase (HDAC) complex rescued the repair defect associated with loss of Esa1 activity, suggesting that NuA4-catalyzed acetylation is important for efficient NER in heterochromatin.     

XRCC1 and base excision repair balance in response to nitric oxide

Inflammation associated reactive oxygen and nitrogen species (RONs), including peroxynitrite (ONOO⁻) and nitric oxide (NO), create base lesions that potentially play a role in the toxicity and large genomic rearrangements associated with many malignancies. Little is known about the role of base excision repair (BER) in removing these endogenous DNA lesions. Here, we explore the role of X-ray repair cross-complementing group 1 (XRCC1) in attenuating RONs-induced genotoxicity. XRCC1 is a scaffold protein critical for BER for which polymorphisms modulate the risk of cancer. We exploited CHO and human glioblastoma cell lines engineered to express varied levels of BER proteins to study XRCC1. Cytotoxicity and the levels of DNA repair intermediates (single-strand breaks; SSB) were evaluated following exposure of the cells to the ONOO⁻ donor, SIN-1, and to gaseous NO. XRCC1 null cells were slightly more sensitive to SIN-1 than wild-type cells. We used small-scale bioreactors to expose cells to NO and found that XRCC1-deficient CHO cells were not sensitive. However, using a molecular beacon assay to test lesion removal in vitro, we found that XRCC1 facilitates AAG-initiated excision of two key NO-induced DNA lesions: 1,N⁶-ethenoadenine and hypoxanthine. Furthermore, overexpression of AAG rendered XRCC1-deficient cells sensitive to NO-induced DNA damage. These results show that AAG is a key glycosylase for BER of NO-induced DNA damage and that XRCC1's role in modulating sensitivity to RONs is dependent upon the cellular level of AAG. This demonstrates the importance of considering the expression of other components of the BER pathway when evaluating the impact of XRCC1 polymorphisms on cancer risk.     

Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N-methyl-N′-nitro-N-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N-methyl-N′-nitro-N-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to γ-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

XRCC1 is required for DNA single-strand break repair in human cells

The X-ray repair cross complementing 1 (XRCC1) protein is required for viability and efficient repair of DNA single-strand breaks (SSBs) in rodents. XRCC1-deficient mouse or hamster cells are hypersensitive to DNA damaging agents generating SSBs and display genetic instability after such DNA damage. The presence of certain polymorphisms in the human XRCC1 gene has been associated with altered cancer risk, but the role of XRCC1 in SSB repair (SSBR) in human cells is poorly defined. To elucidate this role, we used RNA interference to modulate XRCC1 protein levels in human cell lines. A reduction in XRCC1 protein levels resulted in decreased SSBR capacity as measured by the comet assay and intracellular NAD(P)H levels, hypersensitivity to the cell killing effects of the DNA damaging agents methyl methanesulfonate (MMS), hydrogen peroxide and ionizing radiation and enhanced formation of micronuclei following exposure to MMS. Lowered XRCC1 protein levels were also associated with a significant delay in S-phase progression after exposure to MMS. These data clearly demonstrate that XRCC1 is required for efficient SSBR and genomic stability in human cells.     

DNA polymerase beta and flap endonuclease 1 enzymatic specificities sustain DNA synthesis for long patch base excision repair

DNA polymerase beta (pol beta) and flap endonuclease 1 (FEN1) are key players in pol beta-mediated long-patch base excision repair (LP-BER). It was proposed that this type of LP-BER is accomplished through FEN1 removal of a 2- to 11-nucleotide flap created by pol beta strand displacement DNA synthesis. To understand how these enzymes might cooperate during LP-BER, we characterized purified human pol beta DNA synthesis by utilizing various BER intermediates, including single-nucleotide-gapped DNA, nicked DNA, and nicked DNA with various lengths of flaps all with a 5'-terminal tetrahydrofuran (THF) residue. We observed that nicked DNA and nicked-THF flap DNA were poor substrates for pol beta-mediated DNA synthesis; yet, DNA synthesis was strongly stimulated by purified human FEN1. FEN1 did not improve pol beta substrate binding. FEN1 cleavage activity was required for the stimulation, suggesting that FEN1 removed a barrier to pol beta DNA synthesis. In addition, FEN1 cleavage on both nicked and nicked-THF flap DNA resulted in a one-nucleotide gapped DNA molecule that was an ideal substrate for pol beta. This study demonstrates that pol beta cooperates with FEN1 to remove DNA damage via a "Hit and Run" mechanism, involving alternating short gap production by FEN1 and gap filling by pol beta, rather than through coordinated formation and removal of a strand-displaced flap.     

Mapping of the interaction interface of DNA polymerase beta with XRCC1

Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.     

Poly(ADP-ribose) polymerase-1 (PARP-1) is required in murine cell lines for base excision repair of oxidative DNA damage in the absence of DNA polymerase beta

Oxidative DNA base damage is mainly corrected by the base excision repair (BER) pathway, which can be divided into two subpathways depending on the length of the resynthetized patch, either one nucleotide for short patch BER or several nucleotides for long patch BER. The role of proteins in the course of BER processes has been investigated in vitro using purified enzymes and cell-free extracts. In this study, we have investigated the repair of 8-oxo-7,8-dihydroguanine (8-oxoG) in vivo using wild-type, polymerase beta(-/-) (Polbeta(-/-)), poly(ADP-ribose) polymerase-1(-/-) (PARP-1(-/-)), and Polbeta(-/-)PARP-1(-/-) 3T3 cell lines. We used non replicating plasmids containing a 8-oxoG:C base pair to study the repair of the lesion located in a transcribed sequence (TS) or in a non-transcribed sequence (NTS). The results show that 8-oxoG repair in TS is not significantly impaired in cells deficient in Polbeta or PARP-1 or both. Whereas 8-oxoG repair in NTS is normal in Polbeta-null cells, it is delayed in PARP-1-null cells and greatly impaired in cells deficient in both Polbeta and PARP-1. The removal of 8-oxoG and presumably the cleavage at the resulting apurinic/apyrimidinic site are not affected in the PARP-1(-/-)Polbeta(-/-) cell lines. However, 8-oxoG repair is incomplete, yielding plasmid molecules with a nick at the site of the lesion. Therefore, PARP-1(-/-)Polbeta(-/-) cell lines cannot perform 5'-dRP removal and/or DNA repair synthesis. Furthermore, the poly(ADP-ribosyl)ation activity of PARP-1 is essential for 8-oxoG repair in a Polbeta(-/-) context, because expression of the catalytically inactive PARP-1 (E988K) mutant does not restore 8-oxoG repair, whereas an wild type PARP-1 does.