Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7-ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C(Cdh1) through degradation of Cdh1 upon replication block, thereby stabilizing APC/C(Cdh1) substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin-proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.

Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University CZ 775 15 Olomouc Czech Republic;

See more in PubMed

Adams MM, Carpenter PB 2006. Tying the loose ends together in DNA double strand break repair with 53BP1. Cell Div 31: 1–19 PubMed PMC

Albertella MR, Green CM, Lehmann AR, O’Connor MJ 2005. A role for polymerase η in the cellular tolerance to cisplatin-induced damage. Cancer Res 65: 9799–9806 PubMed

Bartek J, Lukas J 2003. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3: 421–429 PubMed

Bartek J, Lukas C, Lukas J 2004. Checking on DNA damage in S phase. Nat Rev Mol Cell Biol 5: 792–804 PubMed

Bassermann F, Frescas D, Guardavaccaro D, Busino L, Peschiaroli A, Pagano M 2008. The Cdc14B–Cdh1–Plk1 axis controls the G2 DNA-damage-response checkpoint. Cell 134: 256–267 PubMed PMC

Blasius M, Forment JV, Thakkar N, Wagner SA, Choudhary C, Jackson SP 2011. A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1. Genome Biol 12: R78. PubMed PMC

Bonte D, Lindvall C, Liu H, Dykema K, Furge K, Weinreich M 2008. Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10: 920–931 PubMed PMC

Chen YW, Cleaver JE, Hanaoka F, Chang CF, Chou KM 2006. A novel role of DNA polymerase η in modulating cellular sensitivity to chemotherapeutic agents. Mol Cancer Res 4: 257–265 PubMed

Cho HJ, Lee EH, Han SH, Chung HJ, Jeong JH, Kwon J, Kim H 2012. Degradation of human RAP80 is cell cycle regulated by Cdc20 and Cdh1 ubiquitin ligases. Mol Cancer Res 10: 615–625 PubMed

Clarke LE, Fountaine TJ, Hennessy J, Bruggeman RD, Clarke JT, Mauger DT, Helm KF 2009. Cdc7 expression in melanomas, Spitz tumors and melanocytic nevi. J Cutan Pathol 36: 433–438 PubMed

Costanzo V, Robertson K, Ying CY, Kim E, Avvedimento E, Gottesman M, Grieco D, Gautier J 2000. Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage. Mol Cell 6: 649–659 PubMed

Costanzo V, Shechter D, Lupardus PJ, Cimprich KA, Gottesman M, Gautier J 2003. An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication. Mol Cell 11: 203–213 PubMed

Day TA, Palle K, Barkley LR, Kakusho N, Zou Y, Tateishi S, Verreault A, Masai H, Vaziri C 2010. Phosphorylated Rad18 directs DNA polymerase η to sites of stalled replication. J Cell Biol 191: 953–966 PubMed PMC

Dierov J, Dierova R, Carroll M 2004. BCR/ABL translocates to the nucleus and disrupts an ATR-dependent intra-S phase checkpoint. Cancer Cell 5: 275–285 PubMed

Dolan WP, Le AH, Schmidt H, Yuan JP, Green M, Forsburg SL 2010. Fission yeast Hsk1 (Cdc7) kinase is required after replication initiation for induced mutagenesis and proper response to DNA alkylation damage. Genetics 185: 39–53 PubMed PMC

Doles J, Oliver TG, Cameron ER, Hsu G, Jacks T, Walker GC, Hemann MT 2010. Suppression of Rev3, the catalytic subunit of Polζ, sensitizes drug-resistant lung tumors to chemotherapy. Proc Natl Acad Sci 107: 20786–20791 PubMed PMC

Ermoli A, Bargiotti A, Brasca MG, Ciavolella A, Colombo N, Fachin G, Isacchi A, Menichincheri M, Molinari A, Montagnoli A, et al. 2009. Cell division cycle 7 kinase inhibitors: 1H-pyrrolo[2,3-b]pyridines, synthesis and structure-activity relationships. J Med Chem 52: 4380–4390 PubMed

Ferreira MF, Santocanale C, Drury LS, Diffley JF 2000. Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex. Mol Cell Biol 20: 242–248 PubMed PMC

Fung AD, Ou J, Bueler S, Brown GW 2002. A conserved domain of Schizosaccharomyces pombe dfp1+ is uniquely required for chromosome stability following alkylation damage during S phase. Mol Cell Biol 22: 4477–4490 PubMed PMC

Gabrielse C, Miller CT, McConnell KH, DeWard A, Fox CA, Weinreich M 2006. A Dbf4p BRCA1 C-terminal-like domain required for the response to replication fork arrest in budding yeast. Genetics 173: 541–555 PubMed PMC

Hess GF, Drong RF, Weiland KL, Slightom JL, Sclafani RA, Hollingsworth RE 1998. A human homolog of the yeast CDC7 gene is overexpressed in some tumors and transformed cell lines. Gene 211: 133–140 PubMed

Hicks JK, Chute CL, Paulsen MT, Ragland RL, Howlett NG, Gueranger Q, Glover TW, Canman CE 2010. Differential roles for DNA polymerases η, ζ, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links. Mol Cell Biol 30: 1217–1230 PubMed PMC

Hishida T, Kubota Y, Carr AM, Iwasaki H 2009. RAD6–RAD18–RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light. Nature 457: 612–615 PubMed

Huang J, Huen MS, Kim H, Leung CC, Glover JN, Yu X, Chen J 2009. RAD18 transmits DNA damage signalling to elicit homologous recombination repair. Nat Cell Biol 11: 592–603 PubMed PMC

Ito S, Taniyami C, Arai N, Masai H 2008. Cdc7 as a potential new target for cancer therapy. Drug News Perspect 21: 481–488 PubMed

Jiang W, Hunter T 1997. Identification and characterization of a human protein kinase related to budding yeast Cdc7p. Proc Natl Acad Sci 94: 14320–14325 PubMed PMC

Jiang W, McDonald D, Hope TJ, Hunter T 1999. Mammalian Cdc7–Dbf4 protein kinase complex is essential for initiation of DNA replication. EMBO J 18: 5703–5713 PubMed PMC

Johnston LH, Masai H, Sugino A 1999. First the CDKs, now the DDKs. Trends Cell Biol 9: 249–252 PubMed

Kim JM, Sato N, Yamada M, Arai K, Masai H 1998. Growth regulation of the expression of mouse cDNA and gene encoding a serine/threonine kinase related to Saccharomyces cerevisiae CDC7 essential for G1/S transition. Structure, chromosomal localization, and expression of mouse gene for S. cerevisiae Cdc7-related kinase. J Biol Chem 273: 23248–23257 PubMed

Kim JM, Nakao K, Nakamura K, Saito I, Katsuki M, Arai K, Masai H 2002. Inactivation of Cdc7 kinase in mouse ES cells results in S-phase arrest and p53-dependent cell death. EMBO J 21: 2168–2179 PubMed PMC

Kim JM, Kakusho N, Yamada M, Kanoh Y, Takemoto N, Masai H 2008. Cdc7 kinase mediates Claspin phosphorylation in DNA replication checkpoint. Oncogene 27: 3475–3482 PubMed

Kulkarni AA, Kingsbury SR, Tudzarova S, Hong HK, Loddo M, Rashid M, Rodriguez-Acebes S, Prevost AT, Ledermann JA, Stoeber K, et al. 2009. Cdc7 kinase is a predictor of survival and a novel therapeutic target in epithelial ovarian carcinoma. Clin Cancer Res 15: 2417–2425 PubMed

Kumagai H, Sato N, Yamada M, Mahony D, Seghezzi W, Lees E, Arai K, Masai H 1999. A novel growth- and cell cycle-regulated protein, ASK, activates human Cdc7-related kinase and is essential for G1/S transition in mammalian cells. Mol Cell Biol 19: 5083–5095 PubMed PMC

Labib K 2010. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Dev 24: 1208–1219 PubMed PMC

Lee AY, Chiba T, Truong LN, Cheng AN, Do J, Cho MJ, Chen L, Wu X 2012. Dbf4 is direct downstream target of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein to regulate intra-S-phase checkpoint. J Biol Chem 287: 2531–2543 PubMed PMC

Listovsky T, Zor A, Laronne A, Brandeis M 2000. Cdk1 is essential for mammalian cyclosome/APC regulation. Exp Cell Res 255: 184–191 PubMed

Listovsky T, Oren YS, Yudkovsky Y, Mahbubani HM, Weiss AM, Lebendiker M, Brandeis M 2004. Mammalian Cdh1/Fzr mediates its own degradation. EMBO J 23: 1619–1626 PubMed PMC

Lukas C, Sorensen CS, Kramer E, Santoni-Rugiu E, Lindeneg C, Peters JM, Bartek J, Lukas J 1999. Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401: 815–818 PubMed

Masai H, Arai K 2000. Dbf4 motifs: Conserved motifs in activation subunits for Cdc7 kinases essential for S-phase. Biochem Biophys Res Commun 275: 228–232 PubMed

Masai H, Arai K 2002. Cdc7 kinase complex: A key regulator in the initiation of DNA replication. J Cell Physiol 190: 287–296 PubMed

Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda M 2010. Eukaryotic chromosome DNA replication: Where, when, and how? Annu Rev Biochem 79: 89–130 PubMed

Mendez J, Stillman B 2000. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: Assembly of prereplication complexes in late mitosis. Mol Cell Biol 20: 8602–8612 PubMed PMC

Montagnoli A, Valsasina B, Croci V, Menichincheri M, Rainoldi S, Marchesi V, Tibolla M, Tenca P, Brotherton D, Albanese C, et al. 2008. A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity. Nat Chem Biol 4: 357–365 PubMed

Nambiar S, Mirmohammadsadegh A, Hassan M, Mota R, Marini A, Alaoui A, Tannapfel A, Hegemann JH, Hengge UR 2007. Identification and functional characterization of ASK/Dbf4, a novel cell survival gene in cutaneous melanoma with prognostic relevance. Carcinogenesis 28: 2501–2510 PubMed

Njagi GD, Kilbey BJ 1982. cdc7-1 a temperature sensitive cell-cycle mutant which interferes with induced mutagenesis in Saccharomyces cerevisiae. Mol Gen Genet 186: 478–481 PubMed

Nojima K, Hochegger H, Saberi A, Fukushima T, Kikuchi K, Yoshimura M, Orelli BJ, Bishop DK, Hirano S, Ohzeki M, et al. 2005. Multiple repair pathways mediate tolerance to chemotherapeutic cross-linking agents in vertebrate cells. Cancer Res 65: 11704–11711 PubMed

Ogino K, Takeda T, Matsui E, Iiyama H, Taniyama C, Arai K, Masai H 2001. Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase. J Biol Chem 276: 31376–31387 PubMed

Pessoa-Brandao L, Sclafani RA 2004. CDC7/DBF4 functions in the translesion synthesis branch of the RAD6 epistasis group in Saccharomyces cerevisiae. Genetics 167: 1597–1610 PubMed PMC

Rendtlew Danielsen JM, Larsen DH, Schou KB, Freire R, Falck J, Bartek J, Lukas J 2009. HCLK2 is required for activity of the DNA damage response kinase ATR. J Biol Chem 284: 4140–4147 PubMed

Saberi A, Hochegger H, Szuts D, Lan L, Yasui A, Sale JE, Taniguchi Y, Murakawa Y, Zeng W, Yokomori K, et al. 2007. RAD18 and poly(ADP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair. Mol Cell Biol 27: 2562–2571 PubMed PMC

Sato N, Arai K, Masai H 1997. Human and Xenopus cDNAs encoding budding yeast Cdc7-related kinases: In vitro phosphorylation of MCM subunits by a putative human homologue of Cdc7. EMBO J 16: 4340–4351 PubMed PMC

Sato N, Sato M, Nakayama M, Saitoh R, Arai K, Masai H 2003. Cell cycle regulation of chromatin binding and nuclear localization of human Cdc7–ASK kinase complex. Genes Cells 8: 451–463 PubMed

Skaar JR, Pagano M 2008. Cdh1: A master G0/G1 regulator. Nat Cell Biol 10: 755–757 PubMed PMC

Sorensen CS, Lukas C, Kramer ER, Peters JM, Bartek J, Lukas J 2000. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis. Mol Cell Biol 20: 7613–7623 PubMed PMC

Sorensen CS, Lukas C, Kramer ER, Peters JM, Bartek J, Lukas J 2001. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex–Cdh1 and cyclin A–Cdk2 during cell cycle progression. Mol Cell Biol 21: 3692–3703 PubMed PMC

Sudo T, Ota Y, Kotani S, Nakao M, Takami Y, Takeda S, Saya H 2001. Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells. EMBO J 20: 6499–6508 PubMed PMC

Syljuasen RG, Sorensen CS, Nylandsted J, Lukas C, Lukas J, Bartek J 2004. Inhibition of Chk1 by CEP-3891 accelerates mitotic nuclear fragmentation in response to ionizing radiation. Cancer Res 64: 9035–9040 PubMed

Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J 2005. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol 25: 3553–3562 PubMed PMC

Szuts D, Simpson LJ, Kabani S, Yamazoe M, Sale JE 2006. Role for RAD18 in homologous recombination in DT40 cells. Mol Cell Biol 26: 8032–8041 PubMed PMC

Tenca P, Brotherton D, Montagnoli A, Rainoldi S, Albanese C, Santocanale C 2007. Cdc7 is an active kinase in human cancer cells undergoing replication stress. J Biol Chem 282: 208–215 PubMed

van Leuken R, Clijsters L, Wolthuis R 2008. To cell cycle, swing the APC/C. Biochim Biophys Acta 1786: 49–59 PubMed

Vanotti E, Amici R, Bargiotti A, Berthelsen J, Bosotti R, Ciavolella A, Cirla A, Cristiani C, D’Alessio R, Forte B, et al. 2008. Cdc7 kinase inhibitors: Pyrrolopyridinones as potential antitumor agents. 1. Synthesis and structure-activity relationships. J Med Chem 51: 487–501 PubMed

Watanabe K, Tateishi S, Kawasuji M, Tsurimoto T, Inoue H, Yamaizumi M 2004. Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination. EMBO J 23: 3886–3896 PubMed PMC

Weinreich M, Stillman B 1999. Cdc7p–Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO J 18: 5334–5346 PubMed PMC

Williams SA, Longerich S, Sung P, Vaziri C, Kupfer GM 2011. The E3 ubiquitin ligase RAD18 regulates ubiquitylation and chromatin loading of FANCD2 and FANCI. Blood 117: 5078–5087 PubMed PMC

Wu X, Lee H 2002. Human Dbf4/ASK promoter is activated through the Sp1 and MluI cell-cycle box (MCB) transcription elements. Oncogene 21: 7786–7796 PubMed

Xie K, Doles J, Hemann MT, Walker GC 2010. Error-prone translesion synthesis mediates acquired chemoresistance. Proc Natl Acad Sci 107: 20792–20797 PubMed PMC

Yamada M, Sato N, Taniyama C, Ohtani K, Arai K, Masai H 2002. A 63-base pair DNA segment containing an Sp1 site but not a canonical E2F site can confer growth-dependent and E2F-mediated transcriptional stimulation of the human ASK gene encoding the regulatory subunit for human Cdc7-related kinase. J Biol Chem 277: 27668–27681 PubMed

Yamashita YM, Okada T, Matsusaka T, Sonoda E, Zhao GY, Araki K, Tateishi S, Yamaizumi M, Takeda S 2002. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J 21: 5558–5566 PubMed PMC

Yamashita N, Kim JM, Koiwai O, Arai K, Masai H 2005. Functional analyses of mouse ASK, an activation subunit for Cdc7 kinase, using conditional ASK knockout ES cells. Genes Cells 10: 551–563 PubMed

Zachariae W, Schwab M, Nasmyth K, Seufert W 1998. Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282: 1721–1724 PubMed

Zegerman P, Diffley JF 2010. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature 467: 474–478 PubMed PMC

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