Abstract

The DNA repair proteins XRCC1 and DNA ligase III are physically associated in human cells and directly interact in vitro and in vivo. Here, we demonstrate that XRCC1 is additionally associated with DNA polymerase-β in human cells and that these polypeptides also directly interact. We also present data suggesting that poly (ADP-ribose) polymerase can interact with XRCC1. Finally, we demonstrate that DNA ligase III shares with poly (ADP-ribose) polymerase the novel function of a molecular DNA nick-sensor, and that the DNA ligase can inhibit activity of the latter polypeptide in vitro. Taken together, these data suggest that the activity of the four polypeptides described above may be co-ordinated in human cells within a single multi-protein complex.

Introduction

Cellular processes of DNA repair are fundamental to the maintenance of genetic integrity and survival, and their inactivation by mutation can dramatically increase individual susceptibility to cancer ( 1–4 ). The use of rodent cell mutants for biochemical studies and for gene cloning has greatly enhanced our understanding of mammalian DNA repair processes in recent years. Two of these mutants, denoted EM9 and EM-C11, are Chinese hamster ovary cell lines that are unable to efficiently rejoin DNA single strand breaks resulting from exposure to agents that induce DNA base-damage, and consequently are hypersensitive to these compounds ( 5–8 ). The human gene that fully corrects the repair defect in EM9 and EM-C11 has been cloned and is denoted XRCC1 ( 6 ). Consistent with its proposed role in single strand break rejoining, XRCC1 polypeptide is physically associated in mammalian cells with DNA ligase III ( 9 , 10 ). On the basis of the cellular phenotype of EM9 and EM-C11, we have proposed that XRCC1 is involved in DNA base excision repair in mammalian cells, at a step subsequent to enzymatic incision of the phosphodiester bond at the damaged nucleoside but prior to or at DNA ligation ( 9 , 10 ). The importance of this polypeptide to mammals is exemplified by the observation that mice in which the XRCC1 locus has been removed by gene targeting are inviable ( 11 ). The biochemical function of XRCC1 is unknown, but the polypeptide most likely fulfils a novel role since it does not exhibit strong homology with any other known protein. Levels of DNA ligase III polypeptide are reduced in EM9 and EM-C11, suggesting that XRCC1 is required for physical stability of the DNA ligase ( 9 , 10 , 12 ). However, physical stabilisation of this protein is unlikely to reflect the primary role of XRCC1. Rather, XRCC1 is most likely required for some aspect of DNA ligase III activity, and/or may interact and affect other components of BER.

The N-terminus of DNA ligase III possesses a putative zinc finger motif that exhibits homology with a ‘nick sensing’ zinc finger present in the nuclear protein, poly (ADP-ribose) polymerase (PARP) ( 13 , 14 ). It is not obvious why DNA ligase III would require a zinc finger since no other known DNA ligase has one, indicating that they are not required for DNA ligation, per se . The zinc finger motif present in PARP confers upon the protein an ability to recognise and rapidly bind DNA containing strand breaks, including those arising during BER ( 15–18 ). Following DNA binding, PARP catalyses the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD + ) to itself and to a number of other nuclear proteins. This process results in the synthesis in large negatively charged poly (ADP-ribose) polymers which facilitate dissociation of the modified PARP protein from DNA, allowing other enzymes to access and repair the DNA strand breaks ( 16 , 17 for recent reviews). The significance of this cyclical DNA binding by PARP is unclear, as is the cellular role of the enzyme. Nevertheless, irrespective of the precise role of PARP, the presence in DNA ligase III of a functional PARP-like ‘nick sensing’ zinc finger would suggest that the activities of these polypeptides are co-ordinated during BER.

In this study, we have begun to address two questions concerning the XRCC1 complex: namely, the identity of any additional polypeptides that associate with XRCC1 and whether the putative zinc finger motif in DNA ligase III does confer ‘nick-sensing’ activity upon this polypeptide.

Materials and Methods

Recombinant human polypeptides

Recombinant human XRCC1 protein containing a C-terminal decahistidine tag (XRCC1-His) was purified by immobilised metal-chelate affinity chromatography (IMAC) from Escherichia coli as described ( 10 ). Recombinant human PARP was expressed in baculovirus and purified as described ( 19 ). Recombinant DNA ligase III was expressed in E.coli from the construct pET16BHL3, which was generated by inserting a Cell II- Dra I fragment isolated from cDNA clone HGS473238 ( 13 ) into the Cell II- Hin dIII site of pET16B (Novagen). To facilitate ligation, recessed 3′ termini produced by Hin dIII were converted to blunt ends with Klenow fragment. The recombinant DNA ligase (His-DNA ligase III) possessed an N-terminal decahistidine tag to facilitate purification by IMAC (as described for XRCC1) and was fully active as judged by its activity on defined oligonucleotide substrates when compared with DNA ligase III partially purified from HeLa nuclear extract (unpublished observations). DNA ligase III polypeptide lacking an intact zinc finger motif (His-DNA ligase III Zn- ) was expressed from the construct pET16BHL3 δ158–170 and purified by IMAC as described for XRCC1. The construct pET16BHL3 δ158–170 was generated from pET16BHL3 by the removal of 39 base pairs (encoding 13 amino acids) with Xma I and Kpn I restriction enzymes and subsequent treatment with T4 DNA polymerase and T4 DNA ligase. Truncated DNA ligase III polypeptide comprised of the first 242 amino acids (His-DNA ligase III 1–242 ), including the putative zinc finger motif, was expressed in E.coli from the construct pET16BHL3 1–242 . His-DNA ligase III 1–242 was purified by IMAC under denaturing conditions (6 M guanidine-HCl), since this polypeptide was largely insoluble in E.coli (unpublished observations). Following purification, His-DNA ligase III 1–242 was renatured by step-wise dialysis in 50 mM Tris-HCl, 0.1 M NaCl, 10% glycerol, 1 mM DTT and decreasing concentrations of guanidine-HCl, with ZnCl 2 included in all but the final step. The construct pET16BHL3 1–242 was generated from pET16BHL3 by digestion with Hin dIII and subsequent religation.That recombinant polypeptides of the expected sizes were expressed and purified was confirmed by SDS-PAGE (data not shown), and protein concentrations determined by a coom-assie blue assay procedure (BioRad).

Escherichia coli harbouring a human DNA polymerase-β expression construct were kindly provided by Dr Sam Wilson, and were grown at 30°C to an OD 600 ∼0.5 after which expression of the DNA polymerase was induced by incubation at 42°C. Aliquots (8 ml) of the culture were sampled prior to and 4 h following induction, and pelleted cells resuspended in 0.5 ml (pre-induction) or 1.5 ml (post-induction) 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM PMSF. After freeze-thawing, cell suspensions were sonicated (6× 15 s bursts on ice; 30 s intervals on ice) and insoluble material pelleted (microfuge, 15 min). Protein concentrations were typically 1.5–2.0 mg/ml.

Antibodies

TL-25 anti-DNA ligase III polyclonal antibodies were raised against His-DNA ligase III, and along with anti-DNA ligase I monoclonal antibody TL-5, was a gift from Tomas Lindahl. Anti-DNA polymerase-β polyclonal antibodies were kind gifts from Sam Wilson. Anti-PARP monoclonal antibodies were raised against recombinant PARP (S. Aoufouchi and C. Milstein, unpublished data), and p450 anti-DNA-PK catalytic subunit (DNA-PK cs ) polyclonal antibodies (31–4) were raised against purified human polypeptide and kindly provided by Steve Jackson. Anti-RPA (p70) and anti-PCNA (PC10) monoclonal antibodies were kind gifts from Rick Wood, and anti-XRCC1 monoclonal antibody 33–2–5 has been described previously ( 10 ).

Oligonucleotides

Oligonucleotides were synthesised by Zeneca Pharmaceuticals and purified by HPLC. To generate oligonucleotide duplexes, complementary oligonucleotides were mixed in equal molar ratios, heated to 70°C for 10 min, and allowed to cool slowly to room temperature to allow annealing. NaCl was added to 50 mM and oligonucleotide duplexes stored at −20°C. Radiolabelled oligonucleotides (2 µg) were prepared, prior to annealing with unlabelled complementary oligonucleotides, by incubation with T4 polynucleotide kinase (25 U) and [γ- 32 P]ATP (40 µCi) in the presence of 10 µM ‘cold’ ATP. Unincorporated nucleotides were removed by sephadex G50 spin-column chromatography.

Mobility shift gel electrophoresis

Radiolabeled oligonucleotide duplexes (0.2 pmol; 10 ng) were incubated with 50–150-fold excess, by weight (0.5–1.5 µg), of either supercoiled plasmid DNA or 12mer oligonucleotide duplex ( Eco RI linker; New England Biolabs) and 5 pmol of either His-DNA ligase III (0.5 µg), His-DNA ligase III Zn- (0.5 µg) or His-DNA ligase III 1–242 (0.18 µg) on ice in 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA. After 10 min, loading buffer was added and samples subjected to non-denaturing polyacrylamide gel electrophoresis (5% gels, BioRad Mini Protean II apparatus) at 20 mA for 2–4 h in pre-chilled 1× TBE, unless otherwise stated. Following electrophoresis, gels were fixed (10% acetic acid, 10 min), dried, and subject to autoradiography.

PARP autoribosylation assays

Recombinant human PARP (0.1 µg; 1 pmol) was incubated in the absence or presence of sonicated calf thymus DNA (40 ng) either with BSA (0.35 µg) and recombinant His-DNA ligase III (0.5 µg; 4.5 pmol), BSA (0.35 µg) and His-XRCC1 (0.35 µg; 4.5 pmol), or with XRCC1-DNA ligase III complex 0.85 µg; 4.5 pmol, pre-formed on ice for 10 min) at room temperature in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 30 µM ZnCl 2 , 2 mM MgCl 2 and [adenylate- 32 P]NAD+ (4 µCi; 8 nmol). After 10 min, reactions were stopped by the addition of SDS-PAGE loading buffer, subjected to SDS-PAGE, and analysed by autoradiography. DNA strand breaks were limiting for PARP activity under the conditions described (results not shown). ATP was omitted from reactions to prevent ATP-dependent DNA ligase activity.

Immunoprecipitation experiments

Anti-XRCC1, PARP, DNA ligase III and RPA antibodies were incubated with 300 µg HeLa crude nuclear extract (CNE; ref. 20 ) in 25 mM Tris-HCl, pH 7.5, 0.2 M KCl, 5% glycerol, 1 mM DTT (300 µl total) for 30 min on ice, after which 30 µl protein-A-coupled Sepharose beads (BioRad) were added and incubation continued for a further 30 min with frequent mixing. After removing 50 µl of the final suspension for samples of CNE prior to immunoprecipitation (‘CNE’), the protein-A-Sepharose beads were pelleted gently in a microfuge (1 s: 3000 r.p.m.) and the immunoprecipitate washed extensively (5× 500 µl) with wash buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM DTT). The immunoprecipitate was washed finally with 1× 100 µl wash buffer, which was kept for analysis (‘wash’ samples), and the immunoprecipitate (‘IP’) resuspended in a further 100 µl of wash buffer. Aliquots of ‘CNE’ samples (5 µl; ∼5 µg total nuclear protein), ‘wash’ samples (10 µl) and ‘IP’ samples (10 µl; protein precipitated from ∼15 µg total nuclear protein) were subject to SDS-PAGE, electroblotted, and immunoblotted with appropriate antibodies. DNA ligase III was detected by immuno-blotting, or as radiolabelled polypeptides following adenylylation in the presence of [α- 32 P]ATP as previously described ( 10 ). Unless indicated, ethidium bromide (10–50 µg/ml) was present in the initial immunoprecipitation reactions to minimise DNA-protein interactions.

Affinity precipitation assays

XRCC1-His (6 µg) or His-DNA ligase III (10 µg) was mixed with E.coli cell extract (75 µg) lacking (pre-induction; see above) or containing (post-induction; see above) human DNA polymerase-β in sonication buffer (see above) containing imidazole (10 mM) and fresh PMSF (1 mM) in a final volume of 220 µl. After 30 min on ice, 25 µl bed volume of NTA-agarose was added to bind histidine-tagged proteins and incubation continued on ice with frequent mixing. After 25 min, a 60 µl sample of the suspension was removed and stored on ice (‘mix’ samples). The NTA-agarose in the remaining reaction mixture was gently pelleted by centrifugation for 1 s in a microfuge at ∼3000 r.p.m. The supernatant containing non-adsorbed material was removed and the NTA-agarose beads washed with 4× 200 µl sonication buffer containing 25 mM imidazole and finally 1× 50 µl sonication buffer containing 25 mM imidazole. The supernatant from the final wash was retained for analysis (‘final wash’). XRCC1-His, His-DNA ligase III and other bound polypeptides were finally eluted from the NTA-agarose beads by 2× 25 µl washes with sonication buffer containing 250 mM imidazole. Aliquots of 5 or 30 µl of ‘mix’ samples, 25 µl of the final wash samples and 25 µl of the combined NTA-agarose eluates were fractionated through 12% SDS-polyacrylamide gels and visualised by coomassie blue staining or immunoblotting, as indicated.

Yeast two-hybrid analysis

XRCC1, PARP, DNA ligase III and DNA polymerase-β open reading frames were subcloned into pACTII and/or pAS1CYH2 vectors (Clontech) to generate chimaeric open reading frames additionally encoding the GAL4 activation domain or the GAL4 DNA binding domain, respectively. These constructs, denoted pAS-XRCC1, pAS-PARP, pAS-Polb, pACT-XRCC1, pACT-PARP and pACT-Lig3, were introduced into Saccharomyces cerevisiae (Y190) either singly or pair-wise and transformants selected on minimal media plates containing tryptophan (for pACTII + , pAS1CYH2 cells) or leucine (for pACTII , pAS1CYH2 + cells), or lacking both (for pACTII + , pAS1CYH2 + cells), as appropriate. Those transformants expressing polypeptides that interacted in this system were identified by the presence of β-galactosidase activity and by their ability to grow on plates lacking histidine (which additionally contained 50 mM 3-aminotriazole to prevent growth resulting from leaky expression of the endogenous histidine gene). To detect β-galactosidase activity, minimal media plates containing independent transformants and, in later experiments, suspensions of individual transformants spotted onto YEPD plates, were lifted onto filter paper (3MM, Whatman), freeze-thawed for 10 s in liquid nitrogen, and incubated at 30°C for 1–16 h on filter paper (3MM) soaked in PBS additionally containing 2 mM MgSO 4 and 2 mg/ml 5-bromo-4-chloro-3-inodoyl-β-D-galactopyranoside (X-Gal, Boehringer Mannheim). To select for loss of pAS constructs, Y190 transformants harbouring appropriate pACT and pAS constructs were grown for multiple generations on YEPD, to facilitate spontaneous loss of plasmid DNA, and replated onto minimal media plates containing tryptophan and cyclohexamide (2.5 µg/ml).

Results

Co-immunoprecipitation of DNA polymerase-β and PARP with XRCC1

We have previously reported that XRCC1 is directly associated in human cells with DNA ligase III ( 9 , 10 , 13 ). To identify other possible polypeptide components of the XRCC1-DNA ligase III complex immunoprecipitates were isolated from HeLa nuclear extract using anti-XRCC1 antibodies, under conditions in which protein-DNA interactions were minimised (see Materials and Methods), and examined for the presence of other polypeptides involved in DNA repair. One polypeptide considered as a possible candidate for associating with XRCC1-DNA ligase III was PARP, since this protein has been implicated in BER. Consistent with this notion, we observed significant co-immunoprecipitation of PARP with XRCC1 and DNA ligase III by anti-XRCC1 antibodies ( Figure 1 , lane 3). Additional experiments revealed that XRCC1, DNA ligase III and PARP were also co-precipitated by anti-DNA ligase III antibodies (lane 6) and anti-PARP antibodies (lane 4), but not by control anti RPA antibodies (lane 5; PARP and DNA ligase III only), suggesting that the polypeptides can associate as a single complex. A second polypeptide considered candidate for associating with the XRCC1 complex was DNA polymerase-β (pol β) since this polypeptide is involved in BER and its activity has been reported to be affected by XRCC1 in vitro (Y. Kubota and T. Lindahl, personal communication). Furthermore, pol β had previously been observed to co-immunoprecipi-tate with PARP from human nuclear extract (S. Aoufouchi, unpublished observations). Consistent with these observations, we observed significant immunoprecipitation of pol β with anti-XRCC1 antibodies ( Figure 1 , lane 3), and complementary experiments revealed that XRCC1 and PARP were immuno-precipitated by anti-pol β antibodies (results not shown).

In contrast with PARP and pol β, a number of other polypeptides failed to co-immunoprecipitate with XRCC1 ( Figure 1 , lane 3), including DNA ligase I, DNA-PK catalytic subunit (‘DNA-PK’), proliferating cell nuclear antigen (‘PCNA’) and the 70 kDa DNA binding subunit of replication protein A (‘RPA’).

Figure 1

Co-immunoprecipitation of XRCC1, DNA ligase III, PARP and pol β from human nuclear extract. The polypeptides present in HeLa nuclear extract prior to immunoprecipitation (CNE, lane 1), the supernatant recovered from the final wash of immunoprecipitated material (Wash, lane 2), and the immuno-precipitates themselves (IP, lanes 3–6) were fractionated by SDS-PAGE and examined for the presence of the polypeptides indicated (left hand side of figure) by immunoblotting. Detection of DNA ligase III as shown was achieved either by immunoblotting (lane 6) or by adenylylation in the presence of [α- 32 P]ATP (lanes 1–5), but similar results were obtained by either method. Immunoprecipitates were generated using antibodies specific for XRCC1 (lane 3), PARP (lane 4), RPA (70 kDa subunit, lane 5) or DNA ligase III (lane 6). ‘ND’ indicates experiment not done.

Figure 1

Co-immunoprecipitation of XRCC1, DNA ligase III, PARP and pol β from human nuclear extract. The polypeptides present in HeLa nuclear extract prior to immunoprecipitation (CNE, lane 1), the supernatant recovered from the final wash of immunoprecipitated material (Wash, lane 2), and the immuno-precipitates themselves (IP, lanes 3–6) were fractionated by SDS-PAGE and examined for the presence of the polypeptides indicated (left hand side of figure) by immunoblotting. Detection of DNA ligase III as shown was achieved either by immunoblotting (lane 6) or by adenylylation in the presence of [α- 32 P]ATP (lanes 1–5), but similar results were obtained by either method. Immunoprecipitates were generated using antibodies specific for XRCC1 (lane 3), PARP (lane 4), RPA (70 kDa subunit, lane 5) or DNA ligase III (lane 6). ‘ND’ indicates experiment not done.

Direct interaction of DNA polymerase-β with XRCC1 in vitro and in vivo

Yeast two-hybrid analysis was employed to identify the interactions responsible for co-immunoprecipitation of pol β with the XRCC1. Neither XRCC1, DNA ligase III, pol β nor PARP polypeptides were themselves able to activate transcription from GAL4 responsive promoters as indicated by the failure of appropriate Y190 transformants to grow in the presence of 3-aminotriazole (3-AT), indicative of histidine auxotrophy, and by the absence of β-galactosidase activity ( Fig. 2 A-D, top panel, and results not shown). However, Y190 cells harbouring both XRCC1 and DNA ligase III fusion proteins exhibited histidine prototrophy (compare Fig. 2 A and B; ‘s-xrcc1: t-lig3’) and contained significant β-galactosidase activity ( Fig. 2 C), consistent with the known interaction of these polypeptides ( 9 , 10 ). In addition, primary Y190 transformants co-expressing XRCC1 and pol β fusion proteins also resulted in histidine prototrophy (compare Fig. 2 A and B; ‘s-polb: t-xrcc1’) and β-galactosidase activity ( Fig. 2 C), indicating that XRCC1 also interacts with DNA polymerase-β. However, we failed to detect any interaction between pol β and either PARP or DNA ligase III in this assay ( Fig. 2 ; ‘s-polb: t-parp’ and ‘s-polb: t-lig3’). To address the specificity of the interaction between XRCC1 and pol β in this assay, we examined XRCC1 for its ability to interact with a number of control polypeptides, including itself. None of the resulting two-hybrid transformants resulted in histidine prototrophy or β-galactosidase activity, confirming that the interaction of XRCC1 with pol β was specific ( Fig. 2 ; ‘s-xrcc1: t-xrcc1’, and results not shown).

Figure 2

XRCC1 can interact directly with DNA polymerase β and possibly PARP in vivo. Suspensions of yeast Y190 cells expressing proteins fused to the GAL4 DNA binding domain (proteins with prefix ‘s-’) or GAL4 activation domain (proteins with prefix ‘t-’) were ‘spotted’ onto appropriate plates (A, B and C, see Materials and Methods) either singly or pairwise as indicated in the key ( D ). Suspensions were grown on appropriate minimal media in the absence of 3-AT ( A ) to allow growth of both histidine prototrophs and auxotrophs, or in the presence of 50 mM 3-AT ( B ) to select for histidine prototrophs, or on YEPD ( C ) for detection of β-galactosidase activity after non-selective growth for 36 h (see Materials and Methods). Histidine prototrophy and β-galactosi-dase activity are indicated by visible circles in (B) and (C), respectively.

Figure 2

XRCC1 can interact directly with DNA polymerase β and possibly PARP in vivo. Suspensions of yeast Y190 cells expressing proteins fused to the GAL4 DNA binding domain (proteins with prefix ‘s-’) or GAL4 activation domain (proteins with prefix ‘t-’) were ‘spotted’ onto appropriate plates (A, B and C, see Materials and Methods) either singly or pairwise as indicated in the key ( D ). Suspensions were grown on appropriate minimal media in the absence of 3-AT ( A ) to allow growth of both histidine prototrophs and auxotrophs, or in the presence of 50 mM 3-AT ( B ) to select for histidine prototrophs, or on YEPD ( C ) for detection of β-galactosidase activity after non-selective growth for 36 h (see Materials and Methods). Histidine prototrophy and β-galactosi-dase activity are indicated by visible circles in (B) and (C), respectively.

To address further the notion that XRCC1 interacts directly with pol β, we examined histidine-tagged human XRCC1 (XRCC1-His) for its ability to bind recombinant human pol β in vitro using an affinity precipitation assay ( 10 , 13 ; Materials and Methods). XRCC1-His was mixed with E.coli cell extract from cells expressing human pol β and subsequently recovered using NTA-agarose beads ( Fig. 3 ). After extensive washing of the NTA-agarose beads, XRCC1-His and associated polypeptides were eluted with 250 mM imidazole and fractionated by SDS-PAGE. Detection of recovered polypeptides by staining with coomassie blue identified two proteins that migrated at the positions expected for XRCC1-His and pol β ( Fig. 3 B, lane 8). That pol b was the smaller of these polypeptides was confirmed by immunoblotting with anti-pol β antibodies ( Fig. 3 C, lane 8). This notion was further supported by the failure of XRCC1-His to recover the latter polypeptide from cell extract prepared from E.coli in which expression of the DNA polymerase was not induced ( Fig. 3 B and C, lane 7). In contrast with XRCC1-His, histidine-tagged DNA ligase III (His-DNA ligase III) failed to recover pol β from E.coli cell extract, indicating that the interaction between XRCC1 and the DNA polymerase was specific ( Fig. 3 B and C, lane 9).

Figure 3

Direct interaction of XRCC1 and DNA polymerase-β in vitro. Histidine-tagged recombinant XRCC1 or DNA ligase III was mixed with NTA-agarose beads and E.coli cell extract lacking or containing pol β for 30 min, and after extensive washing bead-bound proteins were eluted with 250 mM imidazole. ( A ) Escherichia coli cell extracts used in the experiments, either lacking pol β (‘pre-i’; lane 1), or containing pol β (‘post-i’; lane 2). Extracts were prepared from cells prior to or after induction of pol-β expression, respectively. ( B ) Aliquots sampled during the experiment were subjected to SDS-PAGE and gels (12%) stained with coomassie blue. Lanes 1–3: aliquots (30 µl) of initial suspensions (‘mix’ samples) containing NTA-agarose, XRCC1 (lanes 1 and 2; ‘+X’) or DNA ligase III (lane 3; ‘+L3’) and E.coli extract lacking (lane 1) or containing (lanes 2 and 3) pol β. Lanes 4–6: aliquots (25 µl) of the final wash of NTA-agarose beads containing XRCC1 (lanes 4 and 5) or DNA ligase III (lane 6), and associated polypeptides. Lanes 7–9: aliquots (25 µl) of imidazole eluates of NTA-agarose beads containing XRCC1 (lanes 7 and 8; ‘+X’) or DNA ligase III (lane 9; ‘+L3’) and associated polypeptides. Lanes 10 and 11: recombinant XRCC1 marker (0.6 µg) and DNA ligase III marker (2 µg), respectively. ( C ) As ( B ) except that fractionated polypeptides were immunoblotted with anti-pol β antibodies. Arrows indicate the positions of full length XRCC1-His (‘X’; migrates at ∼85 kDa), His-DNA ligase III (‘L3’; migrates at ∼103 kDa) and pol β (‘β’; migrates at ∼39 kDa).

Figure 3

Direct interaction of XRCC1 and DNA polymerase-β in vitro. Histidine-tagged recombinant XRCC1 or DNA ligase III was mixed with NTA-agarose beads and E.coli cell extract lacking or containing pol β for 30 min, and after extensive washing bead-bound proteins were eluted with 250 mM imidazole. ( A ) Escherichia coli cell extracts used in the experiments, either lacking pol β (‘pre-i’; lane 1), or containing pol β (‘post-i’; lane 2). Extracts were prepared from cells prior to or after induction of pol-β expression, respectively. ( B ) Aliquots sampled during the experiment were subjected to SDS-PAGE and gels (12%) stained with coomassie blue. Lanes 1–3: aliquots (30 µl) of initial suspensions (‘mix’ samples) containing NTA-agarose, XRCC1 (lanes 1 and 2; ‘+X’) or DNA ligase III (lane 3; ‘+L3’) and E.coli extract lacking (lane 1) or containing (lanes 2 and 3) pol β. Lanes 4–6: aliquots (25 µl) of the final wash of NTA-agarose beads containing XRCC1 (lanes 4 and 5) or DNA ligase III (lane 6), and associated polypeptides. Lanes 7–9: aliquots (25 µl) of imidazole eluates of NTA-agarose beads containing XRCC1 (lanes 7 and 8; ‘+X’) or DNA ligase III (lane 9; ‘+L3’) and associated polypeptides. Lanes 10 and 11: recombinant XRCC1 marker (0.6 µg) and DNA ligase III marker (2 µg), respectively. ( C ) As ( B ) except that fractionated polypeptides were immunoblotted with anti-pol β antibodies. Arrows indicate the positions of full length XRCC1-His (‘X’; migrates at ∼85 kDa), His-DNA ligase III (‘L3’; migrates at ∼103 kDa) and pol β (‘β’; migrates at ∼39 kDa).

A possible interaction between PARP and XRCC1 in vivo

To dissect the protein interactions responsible for co-immuno-precipitation of PARP with the XRCC1 complex we again utilised the yeast two-hybrid assay. As described above, PARP and pol β did not interact in this system indicating that the co-immunoprecipitation of PARP with the XRCC1 complex was not mediated through these polypeptides. PARP and DNA ligase III similarly failed to interact in the two-hybrid assay, as indicated by the absence of histidine prototrophy and significant β-galactosidase activity in Y190 transformants harbouring the appropriate constructs ( Fig. 2 ; ‘s-parp: t-lig3’). However, PARP and XRCC1 did appear to interact, though in contrast to the interaction of XRCC1 with DNA ligase III and pol β, the interaction between XRCC1 and PARP was not observed in primary transformants unless selected for on plates containing 3-AT. Nevertheless, histidine prototrophic clones selected in this manner contained significant β-galactosidase activity ( Fig. 2 C; ‘s-xrcc1: t-parp’), and subsequent removal of the PAS-XRCC1 plasmid from such clones (see Materials and Methods) resulted in concurrent loss of both phenotypes (results not shown). This indicates that β-galac-tosidase activity and histidine prototrophy were dependent on the presence of the XRCC1 construct and suggests that the apparent interaction was genuine.

Presence of a DNA damage ‘nick sensing’ zinc finger in DNA ligase III

The notion of PARP and DNA ligase III both being present in a multiprotein complex is intriguing, since sequence analysis reveals a putative zinc finger motif near the N-terminus of the DNA ligase that exhibits ∼30% identity with a ‘nick sensing’ zinc finger present in PARP ( 13 ). Furthermore, four of the five basic residues present in PARP that have been suggested to form the interacting face of a DNA binding α-helix are absolutely conserved in DNA ligase III, including the arginine residue essential for the ‘nick-sensing’ activity of PARP ( Fig.4 A and ref. 14 ). The similarity of these two motifs suggests that DNA ligase III may also possess nick-sensing activity. Consistent with this notion is the observed ability of DNA ligase III to inhibit DNA-dependent PARP autoribosylation when added in excess ( Fig. 5 , compare lanes 2, 4 and 5), the simplest explanation for which being that the two polypeptides can compete for DNA binding. To directly address the question of whether DNA ligase III possesses nick-sensing activity we examined the polypeptide for its ability to bind a nicked 70mer oligonucleotide duplex by gel electrophoresis mobility shift assays ( Fig. 6 A). Recombinant histidine-tagged DNA ligase III (His-DNA ligase III) was able to bind the nicked duplex in a manner that was dependent on the single strand break, as indicated by the formation of a well defined mobility shifted complex with this duplex but not with an undamaged control duplex ( Fig. 6 , lanes 3 and 7; top arrow), thus confirming that the polypeptide possesses nick sensing activity. A small amount of faster migrating complex was also formed by His-DNA ligase III which did not reflect nick sensing activity, since it was produced with both nicked and control duplex (lanes 3 and 7, middle arrow). Rather, this complex appears to reflect binding of His-DNA ligase III to DNA double strand breaks since it was not formed when a linear 12mer duplex was used as competitor DNA instead of supercoiled plasmid (results not shown). That the zinc finger motif was responsible for the nick sensing activity of DNA ligase III was suggested by specific binding of the nicked 70mer to a truncated polypeptide comprised of only the first 242 amino acids of the DNA ligase ( Fig. 6 B, lane 2; lower arrow). This polypeptide encodes N-terminal sequences unique to DNA ligase III, as opposed to those sequences present in all ATP-dependent DNA ligases, and includes the zinc finger motif ( Fig. 4 B; His-DNA ligase III 1–242 ). To examine the involvement of the zinc finger motif in DNA binding further, we utilised a mutant DNA ligase III polypeptide lacking an intact zinc finger motif ( Fig. 4 C, His-DNA ligase III Zn- ). The nick sensing abilities of His-DNA ligase III and His-DNA ligase III Zn- were compared under conditions previously found to be relatively stringent for formation of DNA ligase III-DNA complexes, using increased amounts of competitor DNA and electrophoresis at 21°C. Whereas His-DNA ligase III produced a defined mobility-shifted complex under these conditions, albeit at a reduced level, His-DNA ligase III Zn- failed to bind the nicked 70mer ( Fig. 6 C, compare lanes 1 and 2 with 3 and 4). Taken together, these results indicate that the zinc finger motif is responsible for the ‘nick sensing’ activity of DNA ligase III.

Figure 4

DNA ligase III and PARP possess homologous zinc finger motifs. ( A ) Schematic comparing the putative zinc finger motif of DNA ligase III with the 38 residue ‘nick sensing’ zinc finger of PARP. The positions of the putative zinc ion and the co-ordinating cysteine and histidine residues are shown, and were positioned on the basis of those present in PARP. Residues in upper case are conserved between the two polypeptides and those underlined are basic residues which in PARP have been proposed to form the interacting face of a DNA binding α-helix ( 14 ). ( B ) Schematic showing the region (hatched lines) of DNA ligase III present in the truncated polypeptide, DNA ligase III 1–242 . Notice that this polypeptide contains the putative zinc finger domain but none of the conserved motifs (filled boxes) characteristic of all known ATP-depend-ent DNA ligases, as represented here by DNA ligase I (‘I’). ( C ) Schematic showing the 13 amino acids deleted (dotted line) to remove part of the putative zinc finger (residues in upper case) from DNA ligase III, resulting in the polypeptide DNA ligase III Zn- . Residues in upper case are those which form the putative zinc finger, and those in bold upper case are those conserved between DNA ligase III and PARP.

Figure 4

DNA ligase III and PARP possess homologous zinc finger motifs. ( A ) Schematic comparing the putative zinc finger motif of DNA ligase III with the 38 residue ‘nick sensing’ zinc finger of PARP. The positions of the putative zinc ion and the co-ordinating cysteine and histidine residues are shown, and were positioned on the basis of those present in PARP. Residues in upper case are conserved between the two polypeptides and those underlined are basic residues which in PARP have been proposed to form the interacting face of a DNA binding α-helix ( 14 ). ( B ) Schematic showing the region (hatched lines) of DNA ligase III present in the truncated polypeptide, DNA ligase III 1–242 . Notice that this polypeptide contains the putative zinc finger domain but none of the conserved motifs (filled boxes) characteristic of all known ATP-depend-ent DNA ligases, as represented here by DNA ligase I (‘I’). ( C ) Schematic showing the 13 amino acids deleted (dotted line) to remove part of the putative zinc finger (residues in upper case) from DNA ligase III, resulting in the polypeptide DNA ligase III Zn- . Residues in upper case are those which form the putative zinc finger, and those in bold upper case are those conserved between DNA ligase III and PARP.

Figure 5

DNA ligase III can inhibit the autoribosylation activity of PARP in vitro. Recombinant human PARP (1 pmol) was incubated with [adenylate- 32 P]NAD+ in the absence of DNA (lane 1) or in the presence of sonicated calf thymus DNA (40 ng; lanes 2–5) and absence of additional recombinant protein (lane 2), presence of recombinant XRCC1 (4.5 pmol, lane 3), recombinant DNA ligase III (4.5 pmol, lane 4) or XRCC1-DNA ligase III complex (4.5 pmol, lane 6). Following SDS-PAGE, autoribosylated PARP (arrow) was detected by autoradiography.

Figure 5

DNA ligase III can inhibit the autoribosylation activity of PARP in vitro. Recombinant human PARP (1 pmol) was incubated with [adenylate- 32 P]NAD+ in the absence of DNA (lane 1) or in the presence of sonicated calf thymus DNA (40 ng; lanes 2–5) and absence of additional recombinant protein (lane 2), presence of recombinant XRCC1 (4.5 pmol, lane 3), recombinant DNA ligase III (4.5 pmol, lane 4) or XRCC1-DNA ligase III complex (4.5 pmol, lane 6). Following SDS-PAGE, autoribosylated PARP (arrow) was detected by autoradiography.

Discussion

We have previously reported that human XRCC1 and DNA ligase III polypeptides physically interact in vitro and that the two proteins are tightly associated in mammalian cells ( 9 , 10 ). In this report, we demonstrate that XRCC1 also interacts with DNA polymerase-β, in vitro and in vivo , and additionally report a possible interaction between XRCC1 and PARP. Finally, we report that DNA ligase III possesses a novel ‘nick sensing’ zinc finger motif of the type present in PARP, suggesting that this polypeptide may fulfil a unique role unusual to DNA ligases.

XRCC1, DNA ligase III and PARP were co-immunoprecipi-tated from HeLa nuclear extract by antibodies specific for each of the three polypeptides, suggesting that the three polypeptides can associate within a single complex. The observed interaction between XRCC1 and pol β suggests that this polypeptide is also a component of the complex, though the possibility that pol β is in a separate complex with XRCC1 has not been excluded. The notion of co-ordinating polypeptides involved in BER as a multiprotein complex is attractive, since the sequential activity of PARP, pol β and DNA ligase III could fulfil the remaining steps of BER following DNA glycosylase and AP endonuclease activity. However, one caveat to the constitutive presence of PARP within such a complex is the relative abundance of this protein compared with XRCC1, pol β and DNA ligase III. Since PARP is very abundant, much of this polypeptide cannot be constitutively bound to XRCC1 complex. It is possible, therefore, that XRCC1 interacts only with a specific form of PARP, such as that already bound to DNA, for example. The observed requirement for selection on media containing 3-AT for apparent interaction between XRCC1 and PARP in the two-hybrid system could reflect such complexity, since specific mutation or very high levels of expression of one or both polypeptides may be required for significant interaction in the absence of DNA damage. Clearly, however, further work is required to confirm that PARP associates with the XRCC1 complex and that it can interact directly with the latter polypeptide.

The biochemical function of XRCC1 is unclear, but its interaction with pol β in vitro and in vivo is consistent with a role for the polypeptide in BER, since an essential role for the DNA polymerase in this process has already been demonstrated ( 21 , 22 ). One possible function of XRCC1 in this process is as a molecular chaperone, guiding XRCC1 complex to sites of repair by binding to DNA intermediates of BER, for example. Alternatively, XRCC1 may possess an as yet unidentified enzymatic activity, such as modulating the activity of pol β, PARP or DNA ligase III in some way, possibilities that are currently under investigation. Modulation of DNA ligase III activity is the most likely of these possibilities, since XRCC1 is more intimately related to this polypeptide than to pol β or PARP. This notion is suggested by the comparative dependency of the three polypeptides on XRCC1 for physical stability, since cellular levels of the DNA ligase are reduced 3–6-fold in XRCC1 mutants whereas levels of pol β and PARP are unaffected ( 10 ; K. Caldecott, unpublished observations).

Figure 6

DNA ligase III is a molecular ‘nick-sensor’. ( A ) Schematic showing the 70mer oligonucleotide duplex containing a defined single strand break (top duplex), and the 70mer control duplex lacking a single strand break (bottom duplex), used in the experiments below. The positions of the single strand break (arrow) and radioactive 5′ phosphate labels (asterisks) are indicated. ( B ) DNA ligase III and DNA ligase III 1–242 polypeptides specifically affect the electrophoretic mobility of an oligonucleotide containing a defined single strand break. Oligonucleotide duplexes containing (lanes 1–4) or lacking (lanes 5–8) a single strand break were incubated on ice in the absence of recombinant proteins (lanes 1 and 5), or in the presence of either DNA ligase III 1–242 (lanes 2 and 6), DNA ligase III (lanes 3 and 7) or XRCC1 (lanes 4 and 8), prior to electrophoresis in chilled 1× TBE. The positions of complex involving nicked duplex and DNA ligase III 1–242 (bottom arrow) or DNA ligase III (top arrows) are indicated. The position of a non-specific complex involving nicked or undamaged duplex and DNA ligase III is also indicated (middle arrow, lanes 3 and 7). The predominant bands at the bottom of the figure are unbound duplex. Competitor DNA was a 12mer oligonucleotide for experiments involving DNA ligase III 1–242 and supercoiled plasmid for those involving DNA ligase III. ( C ) DNA ligase III Zn- protein lacking an intact zinc finger motif exhibits a reduced ability to bind an oligonucleotide containing a defined single strand break. Nicked oligonucleotide duplex was incubated with either DNA ligase III (lanes 1 and 2) or DNA ligase III Zn- (lanes 3 and 4) in the presence of either 50× (lanes 1 and 3) or 150× (lanes 2 and 4) molar excess of supercoiled competitor DNA, prior to electrophoresis in 1× TBE at room temperature.

Figure 6

DNA ligase III is a molecular ‘nick-sensor’. ( A ) Schematic showing the 70mer oligonucleotide duplex containing a defined single strand break (top duplex), and the 70mer control duplex lacking a single strand break (bottom duplex), used in the experiments below. The positions of the single strand break (arrow) and radioactive 5′ phosphate labels (asterisks) are indicated. ( B ) DNA ligase III and DNA ligase III 1–242 polypeptides specifically affect the electrophoretic mobility of an oligonucleotide containing a defined single strand break. Oligonucleotide duplexes containing (lanes 1–4) or lacking (lanes 5–8) a single strand break were incubated on ice in the absence of recombinant proteins (lanes 1 and 5), or in the presence of either DNA ligase III 1–242 (lanes 2 and 6), DNA ligase III (lanes 3 and 7) or XRCC1 (lanes 4 and 8), prior to electrophoresis in chilled 1× TBE. The positions of complex involving nicked duplex and DNA ligase III 1–242 (bottom arrow) or DNA ligase III (top arrows) are indicated. The position of a non-specific complex involving nicked or undamaged duplex and DNA ligase III is also indicated (middle arrow, lanes 3 and 7). The predominant bands at the bottom of the figure are unbound duplex. Competitor DNA was a 12mer oligonucleotide for experiments involving DNA ligase III 1–242 and supercoiled plasmid for those involving DNA ligase III. ( C ) DNA ligase III Zn- protein lacking an intact zinc finger motif exhibits a reduced ability to bind an oligonucleotide containing a defined single strand break. Nicked oligonucleotide duplex was incubated with either DNA ligase III (lanes 1 and 2) or DNA ligase III Zn- (lanes 3 and 4) in the presence of either 50× (lanes 1 and 3) or 150× (lanes 2 and 4) molar excess of supercoiled competitor DNA, prior to electrophoresis in 1× TBE at room temperature.

Concerning the role of the ‘nick sensing’ zinc finger in DNA ligase III, it is possible that this motif can target the polypeptide, and therefore the XRCC1 complex, to DNA strand breaks arising during BER. It remains to be determined whether DNA ligase III and PARP compete in vitro or in vivo for binding DNA strand breaks, or whether there are subtle differences in specificity and/or affinity for nicked DNA intermediates which prevent direct competition. The observed ability of DNA ligase III to inhibit PARP activity on sonicated calf-thymus DNA is consistent with the former notion, though that the mechanism of this inhibition involves direct competition for DNA breaks requires confirmation. It is also possible that, in vivo , the respective ‘nick-sensing’ activities of these polypeptides are co-ordinated during BER, such that unwanted interference of each polypeptide with the other does not occur.

A multiprotein complex that can conduct BER in vitro was recently identified in testis extract, and contains at least pol b and DNA ligase I ( 23 ). The relationship between this complex and the putative XRCC1 complex described here is unclear at present, though the failure of DNA ligase I to co-immunoprecipitate with XRCC1 ( Fig. 1 ; ref. 10 ) suggests that they are different. It is conceivable that multiple complexes may be involved in BER since the involvement of multiple BER pathways has been proposed in eukaryotic cells, on the basis of the apparent involvement in this process of multiple DNA ligases and DNA polymerases ( 24–26 ). Ultimately, biochemical purification of the XRCC1 complex and detailed analysis of its component polypep-tides is required to fully characterise this structure and to clarify its role in the cellular response to genomic insult.

Acknowledgements

We are indebted to Steve Jackson, Rick Wood and Tomas Lindahl for providing antibodies, and to Sam Wilson for providing pol-β antibodies and cDNA. We also thank John Hickman for support and encouragement. This work was supported by Zeneca Pharmaceuticals and the Medical Research Council (G9603130).

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