ABSTRACT
Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase [alpha]-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase [alpha]-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.
To maintain DNA integrity in dividing cells specific biochemical pathways have evolved to accurately coordinate the cell cycle transitions; these checkpoints link completion of one phase to onset of the following phase (1,3). Moreover, DNA damage arrests the cell cycle and induces a cellular response allowing DNA repair, ensuring high fidelity of genetic information transmission (2).
In eukaryotes the highly conserved DNA polymerase [alpha]-primase complex is responsible for synthesis of short RNA-DNA primers essential for the initiation step of DNA replication. It consists of four distinct subunits: p180 (180 kDa) is the catalytic subunit. The primase is a heterodimer of 48 kDa endowed with catalytic activity; p58 (58 kDa) bears a stimulatory function, p68 (68 kDa) has a tethering function between p180 and the primase (4,5). Components of the replication apparatus may act as sensors of DNA damage to stall replication forks, inducing transcription of DNA damage-inducible genes (6). A defect in the mammalian tumour suppressor gene p53 abrogates G1 arrest in response to ionizing radiation (7,8) by transcriptional activation of genes like GADD45 and p21WAF1, a cyclin-dependent kinase inhibitor (9-11). Furthermore, DNA damage sensors may also transduce the stress signal. ATM, which is mutated in patients with the heritable disorder ataxia telangectasia (AT), induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of ionizing radiation and oxidative damage (12,13). Patients harbouring mutations in p53 or ATM are cancer prone, implicating checkpoint controls in the prevention of genetic instability.
In yeast the catalytic subunit of DNA primase is thought to link the DNA damage response to DNA replication, whilst mutations in the PRIl gene failed to delay bud emergence in response to UV irradiation in G1 (14). Another replication block sensor protein is DNA polymerase [epsilon], which is required for the S -> M checkpoint. Interestingly, adjacent to the location of checkpoint-deficient mutations DNA polymerase [epsilon] encompasses a zinc finger resembling the zinc fingers of PARP involved in binding to single-strand breaks (15), suggesting that both proteins recognize a similar structure in DNA.
Poly(ADP-ribose) polymerase is a component of the immediate cellular response to genotoxic stress, playing a critical role in cell recovery from DNA damage (16,17). Purified PARP was shown to suppress in vitro replication of SV40 DNA (18) and to inhibitDNA replication by human replicative DNA polymerases [alpha], [delta] and [epsilon] (19). In contrast, Simbulan et al. demonstrated that in vitro PARP stimulated DNA polymerase [alpha] through a physical association (20). The same authors demonstrated that the PARP-DNA polymerase [alpha] association was required in differentiation-linked DNA replication (21,22).
Accumulating evidence suggests a permanent or temporary association of PARP with the replication machinery: (i) PARP co-purifies with DNA replication forks (23) and topoisomerase I (24,25); (ii) in vivo PARP modifies replication factors such as RP-A (18) and SV40 T antigen (26); (iii) following exposure to low levels of monofunctional alkylating agents inhibition (16,27) or depletion (28,29) of PARP results invariably in G2/M accumulation, presumably reflecting a failure to complete replication, ultimately leading to a mitotic block. Furthermore, in mice lacking PARP proliferating cells are exquisitely sensitive to DNA damaging agents compared with wild-type cells, as measured by: (i) apoptotic cell death of splenocytes exposed to N-methyl-N-nitrosourea (MNU); (ii) necrosis of the epithelial cells of the small intestine located within the crypts, causing death of PARP-deficient mice by 3 days following 8 Gy [gamma]-irradiation (28). All these data strongly suggest that PARP is a survival factor playing an essential and positive role during DNA damage recovery.
In this work we present evidence that PARP and DNA polymerase [alpha]-primase are physically associated in dividing cells, permitting coordination of the initiation of DNA replication with the resolution of replication blocks induced by DNA strand breaks.
HeLa cells were maintained in DMEM 1000 mg/l glucose medium (Sigma) supplemented with 7% fetal bovine serum (Eurobio) and 0.5% gentamycin (Sigma). Primary fibroblasts (MEFs) were isolated from 13.5 day embryos from homozygous mutant PARP-/- and wild-type PARP +/+ mice as described (28). MEFs were maintained in DMEM 4500 mg/l glucose medium supplemented with 10% fetal bovine serum and 0.5% gentamycin. The cells were grown at 37°C with 5% CO2.
HeLa cells (5 * 106) were washed twice with phosphate-buffered saline (PBS) and lysed on ice in 1 ml lysis buffer (20 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP40, 0.2 mM phenylmethanesulfonyl fluoride and 4 µg/ml each leupeptin, pepstatin and aprotinin). Solubilized cell lysates (200 µg protein), precleared for 16 h with 15 µl protein A-Sepharose beads (Pharmacia Biotech), were incubated for 2 h at 4°C with either monoclonal antibodies [anti-DNA polymerase [alpha]-primase antibodies SJK-132-20 (30), provided by M.Smulson, Georgetown University School of Medicine, Washington, and SJK-237-71 (30), provided by J.Hurwitz, Sloan-Kettering Cancer Center, New York; anti-[beta]-galactosidase clone GAL13, Sigma], a polyclonal anti-PARP antibody or a pre-immune serum. Immunocomplexes were precipitated by addition of 30 µl protein A-Sepharose beads and washed five times in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40 and separated by 10% SDS-PAGE. Proteins were transferred onto nitrocellulose and immunoblotted with appropriate antibodies. Immunoblotting was performed with an enhanced chemiluminescence detection system (Amersham). In in vitro experiments 1 µg purified DNA polymerase [alpha]-primase and the indicated domains of PARP (1 µg each) were pre-incubated in 200 µl lysis buffer for 1 h on ice and immunoprecipitation was performed as described above using the polyclonal anti-PARP antibody or the pre-immune serum as control.
HeLa cells were grown for 24 h on glass coverslips, washed twice with PBS, fixed for 10 min with ice-cold methanol/acetone and washed again with PBS. Cells fixed on coverslips were incubated for 16 h with the first antibodies diluted in PBS, 0.1% Tween, 1% BSA. Dilutions were 1/100 for polyclonal anti-PARP antibody and 1/2 for monoclonal anti-DNA polymerase [alpha] p68 (provided by Dr E.Weiss, ESBS, Illkirch, France). After three washes with PBS containing 0.1% Tween, cells were incubated for 3 h with secondary antibodies (FITC-conjugated anti-rabbit IgG and Texas red-conjugated anti-mouse IgG1 respectively, diluted in PBS 1/200). Observations were made with a confocal microscope equipped with an argon/krypton laser and suitable barrier filters (Leica TCS4D, Heidelberg, Germany).
DNA polymerase activity was tested in 60 µl buffer containing 10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 7.5 mM DTT, 50 µg/ml BSA, 0.5 µg DNase I-activated calf thymus DNA, 25 µM each dATP, dCTP and dGTP, 5 µCi [[alpha]-32P]dCTP (3000 Ci/mmol; Dupont NEN). Incorporation of radiolabelled nucleotides was determined by TCA precipitation. DNA polymerase [alpha]-primase inhibition was performed in the presence of 10 µl SJK 132-20 or an antibody directed against the DNA polymerase [alpha] 68 kDa subunit.
Radiolabelled proteins were produced using the in vitro TNT lysate coupled Transcription-Translation System (Promega) with 30 µCi l-[35S]methionine (1175 Ci/mmol; Dupont NEN).One microgram of purified DNA polymerase [alpha]-primase complex was separated by 8% SDS-PAGE and transferred to nitrocellulose membrane. After renaturation for 16 h at 4°C in 10 ml buffer containing 10 mM Tris-HCl, pH 7.4, 5 mM 2-mercaptoethanol, 0.2% Triton, 0.5% milk powder and 0.25% gelatine the membrane was incubated in 1 ml renaturation buffer containing each radiolabelled polypeptide. The membrane was then washed twice in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween and autoradiographed.
Exponentially growing HeLa cells were maintained for 24 h in medium containing 5 µg/ml aphidicolin (Sigma). Cells were released from the cell cycle block by washing three times with PBS and adding fresh complete medium. At various times after release from the aphidicolin block samples were harvested for immunoprecipitation and flow cytometry analysis using an Epics Elite (Coulter).
Mouse embryonic fibroblasts were synchronized in G0 in DMEM containing 0.1% fetal bovine serum for 96 h. Cells were harvested, treated or mock-treated with 150 µM MMS for 30 min at 37°C and released into fresh complete medium. Twenty four hours later cells were pulse labelled with 10 µM 5-bromodeoxyuridine (BrdU) for 1 h and the percentage of cells in S phase was monitored as described (16).
Previous studies (20) have shown that PARP is physically associated in vitro with DNA polymerase [alpha]. To assess the existence of this association in living cells HeLa whole cell extracts were immunoprecipitated using two different monoclonal antibodies raised against the DNA polymerase [alpha]-primase 180 kDa subunit (30) and the immune complex was subjected to SDS-PAGE. Figure 1A shows that PARP (116 kDa), whose activity was detected by activity blot (31), was immunoprecipitated with a polyclonal anti-PARP antibody (lane 1) and was also specifically co-immunoprecipitated with DNA polymerase [alpha]-primase using anti-DNA polymerase [alpha] antibodies (lanes 2 and 3) but not with an anti-[beta]-galactosidase antibody as a negative control (lane 4).
PARP/DNA polymerase [alpha] intranuclear localization was estimated from optical sections obtained using a confocal laser scanning microscope. Figure 2 shows typical PARP and DNA polymerase patterns of doubly stained nuclei observed in proliferating HeLa cells. Panels A and B show confocal images of DNA polymerase [alpha]-primase and PARP labelling respectively within the same nucleus. Co-localization (yellow) is observed at the nuclear periphery and in nucleoli; both patterns overlapped within the limits of the procedure. Altogether, these results indicate that in vivo the two proteins are in close vicinity and are both preferentially present in the nuclear envelope.
The interacting domains between PARP and DNA polymerase [alpha]-primase were mapped using two independent approaches. First, equimolar amounts of purified homogeneous human DNA polymerase [alpha]-primase and full-length PARP or PARP functional domains (29 kDa DBD or 40 kDa catalytic domain; Fig. 3A) were pre-incubated in vitro on ice. Then an anti-PARP antibody or the pre-immune serum was added to the reaction mixture and the immunoprecipitates were assayed for DNA polymerase [alpha] activity. As shown in Figure 3B, DNA polymerase activity co-immunoprecipitated with full-length PARP as well as with the 29 kDa DBD, but not with the 40 kDa catalytic domain or the pre-immune serum.
To test whether PARP/DNA polymerase [alpha]-primase association is cell cycle regulated HeLa cells were blocked by aphidicolin at the G1/S boundary of the cell cycle; following release cell cycle progression was determined by flow cytometric analysis (Fig. 4A). p180 and PARP were present throughout the cell cycle (Fig. 4B and C respectively). Cell lysates from different stages of the cell cycle were immunoprecipitated with the anti-DNA polymerase [alpha]-primase antibody SJK 132-20. The immune complex was immunoblotted with both monoclonal antibody directed against the 180 kDa subunit of DNA polymerase [alpha]-primase and anti-PARP antibody (Fig. 4D and E respectively). Although DNA polymerase [alpha]-primase was efficiently immunoprecipitated throughout the cell cycle, PARP was found associated with DNA polymerase [alpha]-primase only during the S and G2 phases of the cell cycle, and not during G1 phase.
To evaluate the functional significance of the PARP/DNA polymerase [alpha]-primase interaction total DNA polymerase activity in cellular lysates from embryonic fibroblasts derived from either wild-type PARP+/+ or PARP knock-out mice (PARP-/-) was measured in the presence of nicked DNA. The same amount of the large subunit p180 of DNA polymerase [alpha]-primase was present in both extracts. As shown in Figure 5, DNA polymerase activity was reduced by 50% in PARP-/- cellular lysates compared with lysates obtained from parental PARP+/+ cells, suggesting that PARP may stimulate the active replicative complex in vivo.
The involvement of PARP in progression of the replication fork following DNA damage was monitored by the ability of cells to progress into S phase after exposure to sublethal doses of MMS. MEFs derived from PARP+/+ and PARP-/- mice were synchronized by serum starvation. Immediately following release into fresh medium some cells were exposed to MMS, whereas other cells were left untreated; 24 h later they were all pulse labelled with BrdU (Fig. 6). When untreated only 28% of PARP-/- cells had entered S phase compared with 40% of the parental cells, suggesting that they have been delayed in the cell cycle. Following exposure to a sublethal dose of MMS, progression of PARP+/+ MEFs into S phase was not affected, however, cells lacking PARP showed reduced progression through S phase, since only 21% of cells had entered the S phase. Therefore, PARP-/- cells have a reduced capacity to replicate their genome under DNA damage conditions.
We have shown previously that in proliferating cells in which PARP was inhibited by overproduction of a dominant negative mutant or in PARP-deficient cells sublethal doses of alkylating agents led to G2/M accumulation, whilst the parental cells were able to progress continuously through the cell cycle. Under these DNA damaging conditions sister chromatid exchanges which occur when replication is blocked by unrepaired lesions increased (16,28,35) and cells underwent apoptosis (16,28). These results were tentatively interpreted in the context of a DNA survey mechanism implicating the nick-sensor function of PARP as part of the control of replication fork progression when breaks are present in the template.
In this work we provide evidence that PARP contacts the replication machinery, both in vitro and in vivo: (i) PARP and the DNA polymerase [alpha]-primase tetramer co-immunoprecipitate using antibodies specific for either PARP or DNA polymerase [alpha] and PARP is also immunoprecipitated by an anti-DNA polymerase [beta] antibody (unpublished data), a DNA polymerase specifically involved in base excision repair (36,37); (ii) both proteins are co-localized at the nuclear periphery, known to be enriched in replication enzymes(38) and factors involved in cell cycle regulation and tumour suppression, like Rb (39); (iii) the PARP DNA binding domain (29 kDa) but not the 40 kDa catalytic domain interacts with the catalytic subunit of DNA polymerase [alpha]; (iv) PARP/DNA polymerase [alpha]-primase interaction is cell cycle dependent and occurs during the S and G2 phases; (v) in PARP-deficient fibroblasts DNA polymerase activity is decreased by 30-50% compared with the parental cells in the absence of DNA damage, the rate of ongoing S phase being retarded. In addition, our findings suggest that the function of the DNA binding domain of PARP is more complex than initially thought. Besides its role in nick detection (41) and in transfer of the activation signal to the catalytic domain (42), the PARP DNA binding domain binds to several proteins, e.g. XRCC1 (Masson et al., submitted for publication), histones (43), PARP itself (44) and DNA polymerase [alpha] (this report). PARP/DNA polymerase [alpha]-primase interaction is cell cycle controlled, suggesting that cell cycle-dependent phosphorylation of the p180 or p68 subunits by p34cdc2 kinase might modulate protein-protein interaction (46).
This work was supported by the Deutsche Forschungsgemeinschaft under contracts Wi 319/11-2,5 and Na 190/10-3 (H.P.N.), the CNRS (ACC-SV, radiations ionisantes), the Association pour la Recherche contre la Cancer, Electricité de France, the French Ministère de l'Enseignement Supérieur et de la Recherche (95.V.0015) and the Commissariat à l'Energie Atomique (G.dM). F.D. was supported by the Ligue Nationale Contre le Cancer. We are indebted to C.Waltzinger for FACS analysis and Barbara Windsor for careful reading of the manuscript. We also thank Ulrich Hübscher for the gift of anti-DNA polymerase [delta] antibodies, Jerard Hurwitz for anti-RPA and anti-DNA polymerase [alpha] antibodies, Mark Smulson for anti-DNA polymerase [alpha] antibodies, Juhani Syvaoja for anti-DNA polymerase [epsilon] antibodies, Zdenek Hostomsky for anti-DNA polymerase [beta] antibodies and Etienne Weiss for anti-p68 antibody.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Cell culture
Immunoprecipitation experiments
Confocal laser scanning microscopy
DNA polymerase activity assay
Far western blotting analysis
Cell synchronization
S phase analysis after G1 block release
Results
PARP interacts with DNA polymerase [alpha]-primase
PARP and DNA polymerase [alpha]-primase are co-localized in the HeLa cell nucleus
The DNA binding domain of PARP interacts with the catalytic subunit of DNA polymerase [alpha]-primase
PARP/DNA polymerase [alpha]-primase interaction occurs in a cell cycle-dependent manner
DNA polymerase activity is reduced in cell lysates from PARP-deficient cells derived from PARP knock-out mice
The rate of ongoing S phase is retarded in PARP-deficient cells following DNA damage
Discussion
Acknowledgements
References
REFERENCES
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