p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

The cell cycle inhibitor p21^CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates containing single-strand breaks, we found that full-length recombinant GST-tagged p21 but not a C-terminal domain truncated form of p21 was able to stimulate the PARP-1 binding to BER intermediates with no significant influence on the catalytic activity of PARP-1. In addition, we investigate whether the activation of PARP-1 through poly(ADP-ribose) (PAR) synthesis, is required for its interaction with p21. We have found that in human fibroblasts and in HeLa cells treated with the DNA alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), the interaction of p21 with PARP-1 was greatly dependent on PAR synthesis. In fact, an anti-PAR antibody was able to co-immunoprecipitate p21 and PARP-1 from extracts of MNNG-treated cells, while blocking PAR synthesis with the PARP-1 inhibitor Olaparib, drastically reduced the amount of p21 co-immunoprecipitated by a PARP-1 antibody. Our results provide the first evidence that p21 can stimulate the binding of PARP-1 to DNA repair intermediates, and that this cooperation requires PAR synthesis.     

DNA polymerase β-dependent cell survival independent of XRCC1 expression

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1^+/+ and Xrcc1^−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.     

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21^CDKN1A, thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3′-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21^−/− fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA–p21 dependent manner.     

Interaction of p21(CDKN1A) with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair

The cell-cycle inhibitor p21^CDKN1A has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.     

Repair pathway for PARP-1 DNA-protein crosslinks

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.     

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12–C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1^?/? mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12–C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.     

Human proliferating cell nuclear antigen,poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.     

Poly(ADP-ribose) polymerase-1 modulates DNA repair capacity and prevents formation of DNA double strand breaks

Although poly(ADP-ribose) polymerase-1 (PARP-1) has no enzymatic activity involved in DNA damage processing by the base excision repair (BER) pathway, PARP-1 deficient cells are genetically unstable and sensitive to DNA-damaging agents. To explain this paradox, we investigated the impact of PARP-1 on BER in mammalian cells. We reduced cellular PARP-1 protein levels using siRNA, then introduced DNA damage by hydrogen peroxide treatment and examined the repair response. We find that PARP-1 is not involved in recruitment of the major BER proteins to sites of DNA damage. However, we find that PARP-1 protects excessive DNA single strand breaks (SSBs) from converting into DNA double strand breaks (DSBs) thus preserving them for subsequent repair by BER enzymes. This suggests that PARP-1 plays an important role in BER by extending the ability of BER enzymes to process DNA single strand breaks arising directly after mutagen stress or during processing of DNA lesions following extensive DNA damage.     

Regulatory roles of p21 and apurinic/apyrimidinic endonuclease 1 in base excision repair

Many types of DNA damage induce a cellular response that inhibits replication but allows repair by up-regulating the p53 pathway and inducing p21(Cip1, Waf1, Sdi1). The p21 regulatory protein can bind proliferating cell nuclear antigen (PCNA) and prohibit DNA replication. We show here that p21 also inhibits PCNA stimulation of long patch base excision repair (BER) in vitro. p21 disrupts PCNA-directed stimulation of flap endonuclease 1 (FEN1), DNA ligase I, and DNA polymerase delta. The dilemma is to understand how p21 prevents DNA replication but allows BER in vivo. Differential regulation by p21 is likely to relate to the utilization of DNA polymerase beta, which is not sensitive to p21, in the repair pathway. We have also found that apurinic/apyrimidinic endonuclease 1 (APE1) stimulates long patch BER. Furthermore, neither APE1 activity nor its ability to stimulate long patch BER is significantly affected by p21 in vitro. We propose that APE1 serves as an assembly and coordination factor for long patch BER proteins. APE1 initially cleaves the DNA and then facilitates the sequential binding and catalysis by DNA polymerase beta, DNA polymerase delta, FEN1, and DNA ligase I. This model implies that BER can be regulated differentially, based upon the assembly of relevant proteins around APE1 in the presence or absence of PCNA.     

Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.     

Differential Localisation of PARP-1 N-Terminal Fragment in PARP-1+/+ and PARP-1?/? Murine Cells

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 N-terminal fragment encompassing NLS in PARP-1^+/+ and PARP-1^−/− mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-1^+/+ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-1^−/− cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.     

Poly(ADP-ribose) polymerase-1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts

Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using an in vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair. Inhibition of PARP-1 poly(ADP-ribosyl)ation by 3-aminobenzamide blocks dissociation of PARP-1 from damaged DNA and prevents further repair. We find that excessive poly(ADP-ribosyl)ation occurs when repair intermediates containing single-strand breaks are in excess of the repair capacity of the cell extract, suggesting that repeated binding of PARP-1 to the nicked DNA occurs. We also find increased sensitivity of repair intermediates to nuclease cleavage in PARP-deficient mouse fibroblasts and after depletion of PARP-1 from HeLa whole cell extracts. Our data support the model in which PARP-1 binding to DNA single-strand breaks or repair intermediates plays a protective role when repair is limited.     

DNA Damage Triggers p21WAF1-dependent Emi1 Down-Regulation That Maintains G2 Arrest

Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21^WAF1, a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21^WAF1 in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21^+/+ cells stably arrest in G2, whereas p21^−/− cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21^+/+ cells. Inactivation of APC in irradiated p21^+/+ cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21^−/− cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage.     

Poly(ADP-ribose) polymerase-1 as a regulator of protein-nucleic acid interactions in the processes responding to genotoxic action

Poly(ADP-ribose) polymerase-1 (PARP-1), nuclear protein of higher eukaryotes, specifically detects strand breaks in DNA. When bound to DNA strand breaks, PARP-1 is activated and catalyzes synthesis of poly(ADP-ribose) covalently attached to the row of nuclear proteins, with the main acceptor being PARP-1 itself. This protein participates in a majority of DNA dependent processes: repair, recombination; replication: cell death: apoptosis and necrosis. Poly(ADP-ribosyl)ation of proteins is considered as mechanism, which signals about DNA damage and modulate protein functioning in response to genotoxic impact. The main emphasis is made on the roles of PARP-1 and poly(ADP-ribosyl)ation in base excision repair (BER), the process, which provides repair of DNA breaks. The main proposed functions of PARP-1 in this process are: factor initiating assemblage of protein complex of BER; temporary protection of DNA ends; modulation of chromatin structure via poly(ADP-ribosyl)ation of histones; signaling function in detection of the levels of DNA damage in cell.     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

PCNA-dependent accumulation of CDKN1A into nuclear foci after ionizing irradiation

The cyclin-dependent kinase inhibitor CDKN1A/p21 confers cell-cycle arrest in response to DNA damage and inhibits DNA replication through its direct interaction with the proliferating cell nuclear antigen (PCNA) and cyclin/cyclin-dependent kinase complexes. Previously, we reported that in response to densely ionizing radiation CDKN1A rapidly is recruited to the sites of particle traversal, and that CDKN1A foci formation in response to heavy ions is independent of its transactivation by TP53. Here, we show that exposure of normal human fibroblasts to X-rays or to H2O2 also induces nuclear accumulations of CDKN1A. We find that CDKN1A foci formation in response to radiation damage is dependent on its dephosphorylation and on its direct physical interaction with PCNA. Live cell imaging analyses of ectopically expressed EGFP-CDKN1A and dsRed-PCNA show rapid recruitment of both proteins into foci after radiation damage. Detailed dynamic measurements reveal a slightly delayed recruitment of CDKN1A compared to PCNA, which is best described by bi-exponential curve fitting, taking the preceding binding of PCNA to DNA into account. We propose a regulatory role for CDKN1A in mediating PCNA function after radiation damage, and provide evidence that this role is distinct from its involvement in nucleotide excision repair and unrelated to double-strand break repair.     

Slow accumulation of mutations in Xpc−/− mice upon induction of oxidative stress

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa^?/? and Xpc^?/? exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc^?/? mice, in contrary to Xpa^?/? and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc^?/? mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.