Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

Department of Biochemistry and Molecular Pharmacology New York University School of Medicine New York New York United States of America

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic ; International Clinical Research Centre Centre for Biomolecular and Cellular Engineering Saint Anne's University Hospital Brno Czech Republic

Department of Genetics and Development Columbia University Medical Center New York New York United States of America

Department of Molecular Biology University of Copenhagen Copenhagen N Denmark

International Clinical Research Centre Centre for Biomolecular and Cellular Engineering Saint Anne's University Hospital Brno Czech Republic ; Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

National Centre for Biomolecular Research Masaryk University Brno Czech Republic ; Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic ; International Clinical Research Centre Centre for Biomolecular and Cellular Engineering Saint Anne's University Hospital Brno Czech Republic

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PLoS One. 2014;9(6):e101095 PubMed

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San Filippo J, Sung P, Klein H (2008) Mechanism of Homologous Recombination. Annual Review of Biochemistry 77: 229–257. PubMed

Moynahan ME, Jasin M (2010) Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nature reviews Molecular cell biology 11: 196–207. PubMed PMC

Krejci L, Altmannova V, Spirek M, Zhao X (2012) Homologous recombination and its regulation. Nucleic Acids Res 40: 5795–5818. PubMed PMC

Krogh BO, Symington LS (2004) Recombination proteins in yeast. Annu Rev Genet 38: 233–271. PubMed

Ceballos SJ, Heyer WD (2011) Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination. Biochimica et biophysica acta 1809: 509–523. PubMed PMC

Mazin AV, Mazina OM, Bugreev DV, Rossi MJ (2010) Rad54, the motor of homologous recombination. DNA Repair (Amst) 9: 286–302. PubMed PMC

Sung P, Krejci L, Van Komen S, Sehorn MG (2003) Rad51 recombinase and recombination mediators. J Biol Chem 278: 42729–42732. PubMed

Agarwal S, van Cappellen WA, Guenole A, Eppink B, Linsen SE, et al. (2011) ATP-dependent and independent functions of Rad54 in genome maintenance. J Cell Biol 192: 735–750. PubMed PMC

Wolner B, Peterson CL (2005) ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break. J Biol Chem 280: 10855–10860. PubMed

Mazin AV, Alexeev AA, Kowalczykowski SC (2003) A novel function of Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J Biol Chem 278: 14029–14036. PubMed

Clever B, Interthal H, Schmuckli-Maurer J, King J, Sigrist M, et al. (1997) Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins. EMBO J 16: 2535–2544. PubMed PMC

Jiang H, Xie Y, Houston P, Stemke-Hale K, Mortensen UH, et al. (1996) Direct association between the yeast Rad51 and Rad54 recombination proteins. J Biol Chem 271: 33181–33186. PubMed

Raschle M, Van Komen S, Chi P, Ellenberger T, Sung P (2004) Multiple interactions with the Rad51 recombinase govern the homologous recombination function of Rad54. J Biol Chem 279: 51973–51980. PubMed

Alexeev A, Mazin A, Kowalczykowski SC (2003) Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. Nat Struct Biol 10: 182–186. PubMed

Jaskelioff M, Van Komen S, Krebs JE, Sung P, Peterson CL (2003) Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J Biol Chem 278: 9212–9218. PubMed

Petukhova G, Stratton S, Sung P (1998) Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393: 91–94. PubMed

Petukhova G, Stratton SA, Sung P (1999) Single strand DNA binding and annealing activities in the yeast recombination factor Rad59. J Biol Chem 274: 33839–33842. PubMed

Ristic D, Wyman C, Paulusma C, Kanaar R (2001) The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc Natl Acad Sci U S A 98: 8454–8460. PubMed PMC

Van Komen S, Petukhova G, Sigurdsson S, Stratton S, Sung P (2000) Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol Cell 6: 563–572. PubMed

Li X, Heyer WD (2009) RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae . Nucleic Acids Res 37: 638–646. PubMed PMC

Amitani I, Baskin RJ, Kowalczykowski SC (2006) Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 23: 143–148. PubMed

Solinger JA, Heyer WD (2001) Rad54 protein stimulates the postsynaptic phase of Rad51 protein-mediated DNA strand exchange. Proc Natl Acad Sci U S A 98: 8447–8453. PubMed PMC

Solinger JA, Kiianitsa K, Heyer WD (2002) Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10: 1175–1188. PubMed

Bugreev DV, Mazina OM, Mazin AV (2006) Rad54 protein promotes branch migration of Holliday junctions. Nature 442: 590–593. PubMed

Maloisel L, Bhargava J, Roeder GS (2004) A role for DNA polymerase delta in gene conversion and crossing over during meiosis in Saccharomyces cerevisiae. Genetics 167: 1133–1142. PubMed PMC

Maloisel L, Fabre F, Gangloff S (2008) DNA polymerase delta is preferentially recruited during homologous recombination to promote heteroduplex DNA extension. Mol Cell Biol 28: 1373–1382. PubMed PMC

Sebesta M, Burkovics P, Haracska L, Krejci L (2011) Reconstitution of DNA repair synthesis in vitro and the role of polymerase and helicase activities. DNA Repair (Amst) 10: 567–576. PubMed PMC

Li X, Stith CM, Burgers PM, Heyer W-D (2009) PCNA Is Required for Initiation of Recombination- Associated DNA Synthesis by DNA Polymerase &delta. Molecular Cell 36: 704–713. PubMed PMC

Matulova P, Marini V, Burgess RC, Sisakova A, Kwon Y, et al. (2009) Co-operativity of Mus81-Mms4 with Rad54 in the resolution of recombination and replication intermediates. J Biol Chem. 284: 7733–7745. PubMed PMC

Mazina OM, Mazin AV (2008) Human Rad54 protein stimulates human Mus81-Eme1 endonuclease. Proc Natl Acad Sci U S A 105: 18249–18254. PubMed PMC

Klein HL (2001) Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Δ with other DNA repair genes in Saccharomyces cerevisiae . Genetics 157: 557–565. PubMed PMC

Palladino F, Klein HL (1992) Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants. Genetics 132: 23–37. PubMed PMC

Sugawara N, Wang X, Haber JE (2003) In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol Cell 12: 209–219. PubMed

Warbrick E (2000) The puzzle of PCNA's many partners. Bioessays 22: 997–1006. PubMed

Moldovan GL, Pfander B, Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129: 665–679. PubMed

Gilljam KM, Feyzi E, Aas PA, Sousa MM, Muller R, et al. (2009) Identification of a novel, widespread, and functionally important PCNA-binding motif. J Cell Biol 186: 645–654. PubMed PMC

Bruning JB, Shamoo Y (2004) Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. Structure 12: 2209–2219. PubMed

Gulbis JM, Kelman Z, Hurwitz J, O'Donnell M, Kuriyan J (1996) Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell 87: 297–306. PubMed

Freudenthal BD, Gakhar L, Ramaswamy S, Washington MT (2010) Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange. Nature structural & molecular biology 17: 479–484. PubMed PMC

Ayyagari R, Impellizzeri KJ, Yoder BL, Gary SL, Burgers PM (1995) A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol Cell Biol 15: 4420–4429. PubMed PMC

Eissenberg JC, Ayyagari R, Gomes XV, Burgers PM (1997) Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon. Mol Cell Biol 17: 6367–6378. PubMed PMC

Balajee AS, Geard CR (2001) Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells. Nucleic Acids Res 29: 1341–1351. PubMed PMC

Moggs JG, Grandi P, Quivy JP, Jonsson ZO, Hubscher U, et al. (2000) A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage. Mol Cell Biol 20: 1206–1218. PubMed PMC

Gomes XV, Burgers PM (2000) Two modes of FEN1 binding to PCNA regulated by DNA. Embo J 19: 3811–3821. PubMed PMC

Armstrong AA, Mohideen F, Lima CD (2012) Recognition of SUMO-modified PCNA requires tandem receptor motifs in Srs2. Nature 482: 59–63. PubMed PMC

Kolesar P, Sarangi P, Altmannova V, Zhao X, Krejci L (2012) Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation. Nucleic Acids Research 40: 7831–7843. PubMed PMC

Johansson E, Garg P, Burgers PM (2004) The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding. J Biol Chem 279: 1907–1915. PubMed

Game JC, Mortimer RK (1974) A genetic study of x-ray sensitive mutants in yeast. Mutat Res 24: 281–292. PubMed

Arbel A, Zenvirth D, Simchen G (1999) Sister chromatid-based DNA repair is mediated by RAD54, not by DMC1 or TID1 . EMBO J 18: 2648–2658. PubMed PMC

Shinohara M, Shita-Yamaguchi E, Buerstedde JM, Shinagawa H, Ogawa H, et al. (1997) Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis. Genetics 147: 1545–1556. PubMed PMC

Aguilera A, Klein HL (1988) Genetic control of intrachromosomal recombination in Saccharomyces cerevisiae. I. Isolation and genetic characterization of hyper-recombination mutations. Genetics 119: 779–790. PubMed PMC

Fabre F, Chan A, Heyer WD, Gangloff S (2002) Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc Natl Acad Sci U S A 99: 16887–16892. PubMed PMC

Petukhova G, Van Komen S, Vergano S, Klein H, Sung P (1999) Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation. J Biol Chem 274: 29453–29462. PubMed

Burkovics P, Sebesta M, Sisakova A, Plault N, Szukacsov V, et al. (2013) Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis. Embo J 32: 742–755. PubMed PMC

Pfander B, Moldovan G-L, Sacher M, Hoege C, Jentsch S (2005) SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436: 428–433. PubMed

Ulrich HD (2007) PCNA-SUMO and Srs2: a model SUMO substrate-effector pair. Biochemical Society transactions 35: 1385–1388. PubMed

Sherman F (1991) Getting started with yeast. Methods in Enzymology 194: 3–21. PubMed

Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630. PubMed

Zhao X, Muller EGD, Rothstein R (1998) A Suppressor of Two Essential Checkpoint Genes Identifies a Novel Protein that Negatively Affects dNTP Pools. Mol. Cell 2: 329–340. PubMed

Erdeniz N, Mortensen UH, Rothstein R (1997) Cloning-free PCR-based allele replacement methods. Genome Res 7: 1174–1183. PubMed PMC

Smirnova M, Van Komen S, Sung P, Klein HL (2004) Effects of tumor-associated mutations on Rad54 functions. J Biol Chem 279: 24081–24088. PubMed

Bernstein KA, Shor E, Sunjevaric I, Fumasoni M, Burgess RC, et al. (2009) Sgs1 function in the repair of DNA replication intermediates is separable from its role in homologous recombinational repair. EMBO J. 28: 915–925. PubMed PMC

Burgess RC, Rahman S, Lisby M, Rothstein R, Zhao X (2007) The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Molecular and Cellular Biology 27: 6153–6162. PubMed PMC

Krejci L, Van Komen S, Li Y, Villemain J, Reddy MS, et al. (2003) DNA helicase Srs2 disrupts the Rad51 presynaptic filament. Nature 423: 305–309. PubMed

Sugawara N, Haber JE (2006) Repair of DNA double strand breaks: in vivo biochemistry. Methods in Enzymology 408: 416–429. PubMed

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I