Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.

Department of Genome Dynamics Institute of Molecular Genetics of the Czech Academy of Sciences 142 20 Prague 4 Czech Republic

Department of Radiation Genetics Graduate School of Medicine Kyoto University 606 8501 Kyoto Japan

Genome Damage and Stability Centre School of Life Sciences University of Sussex Falmer Brighton BN1 9RQ UK

Institute of Biochemistry of the Swiss Federal Institute of Technology Otto Stern Weg 3 8093 Zurich Switzerland

Institute of Molecular Cancer Research of the University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland

Institute of Molecular Life Sciences of the University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland

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Scholes D.T., Banerjee M., Bowen B., Curcio M.J.. Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance. Genetics. 2001; 159:1449–1465. PubMed PMC

Bellaoui M., Chang M., Ou J., Xu H., Boone C., Brown G.W.. Elg1 forms an alternative RFC complex important for DNA replication and genome integrity. EMBO J. 2003; 22:4304–4313. PubMed PMC

Kanellis P., Agyei R., Durocher D.. Elg1 forms an alternative PCNA-interacting RFC complex required to maintain genome stability. Curr. Biol. 2003; 13:1583–1595. PubMed

Kaliraman V., Mullen J.R., Fricke W.M., Bastin-Shanower S.A., Brill S.J.. Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. Genes Dev. 2001; 15:2730–2740. PubMed PMC

Chen C., Umezu K., Kolodner R.D.. Chromosomal rearrangements occur in S. cerevisiae rfa1 mutator mutants due to mutagenic lesions processed by double-strand-break repair. Mol. Cell. 1998; 2:9–22. PubMed

Mossi R., Hubscher U.. Clamping down on clamps and clamp loaders–the eukaryotic replication factor C. Eur. J. Biochem. 1998; 254:209–216. PubMed

Yao N.Y., O’Donnell M.. The RFC clamp loader: structure and function. Subcell. Biochem. 2012; 62:259–279. PubMed PMC

Friedberg E.C., Aguilera A., Gellert M., Hanawalt P.C., Hays J.B., Lehmann A.R., Lindahl T., Lowndes N., Sarasin A., Wood R.D.. DNA repair: from molecular mechanism to human disease. DNA Repair (Amst.). 2006; 5:986–996. PubMed

Yao N., Turner J., Kelman Z., Stukenberg P.T., Dean F., Shechter D., Pan Z.Q., Hurwitz J., O’Donnell M.. Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases. Genes Cells. 1996; 1:101–113. PubMed

Yao N.Y., Johnson A., Bowman G.D., Kuriyan J., O’Donnell M.. Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C. J. Biol. Chem. 2006; 281:17528–17539. PubMed

Kubota T., Nishimura K., Kanemaki M.T., Donaldson A.D.. The Elg1 replication factor C-like complex functions in PCNA unloading during DNA replication. Mol. Cell. 2013; 50:273–280. PubMed

Naiki T., Kondo T., Nakada D., Matsumoto K., Sugimoto K.. Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway. Mol. Cell. Biol. 2001; 21:5838–5845. PubMed PMC

Majka J., Burgers P.M.. Yeast Rad17/Mec3/Ddc1: a sliding clamp for the DNA damage checkpoint. Proc. Natl Acad. Sci. U.S.A. 2003; 100:2249–2254. PubMed PMC

Mayer M.L., Gygi S.P., Aebersold R., Hieter P.. Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae. Mol. Cell. 2001; 7:959–970. PubMed

Parnas O., Zipin-Roitman A., Mazor Y., Liefshitz B., Ben-Aroya S., Kupiec M.. The ELG1 clamp loader plays a role in sister chromatid cohesion. PLoS One. 2009; 4:e5497. PubMed PMC

Parnas O., Zipin-Roitman A., Pfander B., Liefshitz B., Mazor Y., Ben-Aroya S., Jentsch S., Kupiec M.. Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA. EMBO J. 2010; 29:2611–2622. PubMed PMC

Parnas O., Amishay R., Liefshitz B., Zipin-Roitman A., Kupiec M.. Elg1, the major subunit of an alternative RFC complex, interacts with SUMO-processing proteins. Cell Cycle. 2011; 10:2894–2903. PubMed

Hoege C., Pfander B., Moldovan G.L., Pyrowolakis G., Jentsch S.. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 2002; 419:135–141. PubMed

Johnson C., Gali V.K., Takahashi T.S., Kubota T.. PCNA retention on DNA into G2/M phase causes genome instability in cells lacking Elg1. Cell Rep. 2016; 16:684–695. PubMed PMC

Aroya S.B., Kupiec M.. The Elg1 replication factor C-like complex: a novel guardian of genome stability. DNA Repair (Amst.). 2005; 4:409–417. PubMed

Gazy I., Liefshitz B., Parnas O., Kupiec M.. Elg1, a central player in genome stability. Mutat Res Rev Mutat Res. 2015; 763:267–279. PubMed

Sikdar N., Banerjee S., Lee K.Y., Wincovitch S., Pak E., Nakanishi K., Jasin M., Dutra A., Myung K.. DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity. Cell Cycle. 2009; 8:3199–3207. PubMed PMC

Lee K.Y., Yang K., Cohn M.A., Sikdar N., D’Andrea A.D., Myung K.. Human ELG1 regulates the level of ubiquitinated proliferating cell nuclear antigen (PCNA) through Its interactions with PCNA and USP1. J. Biol. Chem. 2010; 285:10362–10369. PubMed PMC

Kang M.S., Ryu E., Lee S.W., Park J., Ha N.Y., Ra J.S., Kim Y.J., Kim J., Abdel-Rahman M., Park S.H. et al. .. Regulation of PCNA cycling on replicating DNA by RFC and RFC-like complexes. Nat. Commun. 2019; 10:2420. PubMed PMC

Bajrami I., Frankum J.R., Konde A., Miller R.E., Rehman F.L., Brough R., Campbell J., Sims D., Rafiq R., Hooper S. et al. .. Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Cancer Res. 2014; 74:287–297. PubMed PMC

Maleva Kostovska I., Wang J., Bogdanova N., Schurmann P., Bhuju S., Geffers R., Durst M., Liebrich C., Klapdor R., Christiansen H. et al. .. Rare ATAD5 missense variants in breast and ovarian cancer patients. Cancer Lett. 2016; 376:173–177. PubMed

Bell D.W., Sikdar N., Lee K.Y., Price J.C., Chatterjee R., Park H.D., Fox J., Ishiai M., Rudd M.L., Pollock L.M. et al. .. Predisposition to cancer caused by genetic and functional defects of mammalian Atad5. PLoS Genet. 2011; 7:e1002245. PubMed PMC

Kohzaki M., Nishihara K., Hirota K., Sonoda E., Yoshimura M., Ekino S., Butler J.E., Watanabe M., Halazonetis T.D., Takeda S.. DNA polymerases nu and theta are required for efficient immunoglobulin V gene diversification in chicken. J. Cell Biol. 2010; 189:1117–1127. PubMed PMC

Strom C.E., Johansson F., Uhlen M., Szigyarto C.A., Erixon K., Helleday T.. Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate. Nucleic Acids Res. 2011; 39:3166–3175. PubMed PMC

Lee K.Y., Fu H., Aladjem M.I., Myung K.. ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin. J. Cell Biol. 2013; 200:31–44. PubMed PMC

Wyatt M.D., Pittman D.L.. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks. Chem. Res. Toxicol. 2006; 19:1580–1594. PubMed PMC

Beranek D.T. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat. Res. 1990; 231:11–30. PubMed

Lundin C., North M., Erixon K., Walters K., Jenssen D., Goldman A.S., Helleday T.. Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Res. 2005; 33:3799–3811. PubMed PMC

Fu D., Calvo J.A., Samson L.D.. Balancing repair and tolerance of DNA damage caused by alkylating agents. Nat. Rev. Cancer. 2012; 12:104–120. PubMed PMC

Hecht S.M. Bleomycin: New perspectives on the mechanism of action. J. Nat. Prod. 2000; 63:158–168. PubMed

Podust L.M., Podust V.N., Sogo J.M., Hubscher U.. Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA. Mol. Cell. Biol. 1995; 15:3072–3081. PubMed PMC

Tomasz M. Mitomycin C: small, fast and deadly (but very selective). Chem. Biol. 1995; 2:575–579. PubMed

Moldovan G.L., D’Andrea A.D.. How the fanconi anemia pathway guards the genome. Annu. Rev. Genet. 2009; 43:223–249. PubMed PMC

Wessel S.R., Mohni K.N., Luzwick J.W., Dungrawala H., Cortez D.. Functional analysis of the replication fork proteome identifies BET proteins as PCNA regulators. Cell Rep. 2019; 28:3497–3509. PubMed PMC

Kubota T., Myung K., Donaldson A.D.. Is PCNA unloading the central function of the Elg1/ATAD5 replication factor C-like complex. Cell Cycle. 2013; 12:2570–2579. PubMed PMC

Bryant H.E., Schultz N., Thomas H.D., Parker K.M., Flower D., Lopez E., Kyle S., Meuth M., Curtin N.J., Helleday T.. Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature. 2005; 434:913–917. PubMed

Farmer H., McCabe N., Lord C.J., Tutt A.N., Johnson D.A., Richardson T.B., Santarosa M., Dillon K.J., Hickson I., Knights C. et al. .. Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005; 434:917–921. PubMed

D’Andrea A.D. Mechanisms of PARP inhibitor sensitivity and resistance. DNA Repair (Amst.). 2018; 71:172–176. PubMed

Wang Y., Luo W., Wang Y.. PARP-1 and its associated nucleases in DNA damage response. DNA Repair (Amst.). 2019; 81:102651. PubMed PMC

Hanzlikova H., Kalasova I., Demin A.A., Pennicott L.E., Cihlarova Z., Caldecott K.W.. The importance of Poly(ADP-Ribose) polymerase as a sensor of unligated okazaki fragments during DNA replication. Mol. Cell. 2018; 71:319–331. PubMed PMC

Murai J., Huang S.Y., Das B.B., Renaud A., Zhang Y., Doroshow J.H., Ji J., Takeda S., Pommier Y.. Trapping of PARP1 and PARP2 by clinical PARP inhibitors. Cancer Res. 2012; 72:5588–5599. PubMed PMC

Murai J., Huang S.Y., Renaud A., Zhang Y., Ji J., Takeda S., Morris J., Teicher B., Doroshow J.H., Pommier Y.. Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib. Mol. Cancer Ther. 2014; 13:433–443. PubMed PMC

Kubota T., Katou Y., Nakato R., Shirahige K., Donaldson A.D.. Replication-Coupled PCNA unloading by the Elg1 complex occurs Genome-wide and requires okazaki fragment ligation. Cell Rep. 2015; 12:774–787. PubMed PMC

Gali V.K., Dickerson D., Katou Y., Fujiki K., Shirahige K., Owen-Hughes T., Kubota T., Donaldson A.D.. Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation. PLos Genet. 2018; 14:e1007783. PubMed PMC

Giovannini S., Weller M.C., Repmann S., Moch H., Jiricny J.. Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage. Nucleic Acids Res. 2019; 47:9132–9143. PubMed PMC

Markkanen E. Not breathing is not an option: How to deal with oxidative DNA damage. DNA Repair (Amst.). 2017; 59:82–105. PubMed

Lia D., Reyes A., de Melo Campos J.T.A., Piolot T., Baijer J., Radicella J.P., Campalans A.. Mitochondrial maintenance under oxidative stress depends on mitochondrially localised α-OGG1. J. Cell Sci. 2018; 131:jcs213538. PubMed

Park S.H., Kang N., Song E., Wie M., Lee E.A., Hwang S., Lee D., Ra J.S., Park I.B., Park J. et al. .. ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress. Nat. Commun. 2019; 10:5718. PubMed PMC

PARP inhibition impedes the maturation of nascent DNA strands during DNA replication