LETTERS Single-stranded DNA-binding protein hSSB1 is critical for genomic stability Derek J. Richard 1 , Emma Bolderson 1 , Liza Cubeddu 2,3 , Ross I. M. Wadsworth 2 , Kienan Savage 1,4 , Girdhar G. Sharma 5 , Matthew L. Nicolette 6 , Sergie Tsvetanov 1 , Michael J. McIlwraith 7 , Raj K. Pandita 5 , Shunichi Takeda 8 , Ronald T. Hay 9 , Jean Gautier 10 , Stephen C. West 7 , Tanya T. Paull 6 , Tej K. Pandita 5 , Malcolm F. White 2 & Kum Kum Khanna 1 Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquit- ous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detec- tion and repair 1 . SSBs have multiple roles in binding and seques- tering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer 1 . Here we describe a second human SSB (hSSB1), with a domain organi- zation closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co- localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse end- points in the cellular DNA damage response. Ionizing radiation and anti-cancer drugs can induce DNA DSBs, which are highly cytotoxic lesions. In the S and G2 phases of the cell cycle, homologous recombination can be used to repair DSBs. To initiate homologous recombination, DNA is resected and then bound by RPA, a eukaryotic SSB, to facilitate Rad51 nucleofilament formation and strand invasion 2 . Here we show that, in addition to RPA, the human genome encodes two further conserved SSB homo- logues, present on chromosomes 12q13.3 and 2q32.3, which we have named hSSB1 and hSSB2, respectively. The main focus of this study, hSSB1, is highly represented in EST libraries from a variety of tissues. It is conserved in metazoa, comprising an amino-terminal oligonucleotide/oligosaccharide-binding-fold domain, followed by a more divergent carboxy-terminal domain (Supplementary Fig. 1). Like RPA, recombinant hSSB1 binds specifically to ssDNA substrates (Fig. 1a and Supplementary Fig. 2), in particular to 1 Signal Transduction Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia. 2 Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK. 3 School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia. 4 Central Clinical Division, School of Medicine, University of Queensland, Queensland 4072, Australia. 5 Department of Radiation Oncology, Washington University School of Medicine, St Louis, Missouri 63108, USA. 6 Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712, USA. 7 London Research Institute, Clare Hall Laboratories, Cancer Research UK, South Mimms, Hertfordshire EN6 3LD, U.K. 8 Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan. 9 Division of Gene Regulation and Expression, Wellcome Biocentre, University of Dundee, Dundee DD1 5EH, U.K. 10 Institute for Cancer Genetics, Columbia University Medical Center, New York, New York 10032, USA. (h) 0 0.5 1 1.5 2 3 Actin hSSB1 Ultraviolet Actin Ionizing rad. hSSB1 b + + + + – ATM – + – + – Ionizing rad. hSSB1 T117A hSSB1 ATM hSSB1 d e – + – + Ionizing rad. C3ABR L3 ATM hSSB1 [ 32 P]hSSB1 [ 32 P]hSSB1 a c – + – + IR Actin hSSB1 ATM sicontrol siATM Bound DNA Free DNA hSSB1 Figure 1 | ATM-dependent stabilization and phosphorylation of hSSB1 after ionizing radiation. a, Electrophoretic mobility shift analysis showing binding of recombinant hSSB1 to ssDNA substrates, d30T (top), a synthetic replication fork (middle) and dsDNA (bottom). The radiolabel is marked with a black circle. b, Immunoblots of hSSB1 using cell extracts from neonatal foreskin fibroblasts (NFFs) exposed to ionizing (6 Gy) or ultraviolet (20 J m 22 ) radiation. Cells were harvested at the indicated time points and immunoblotted for hSSB1. c, Western blots of hSSB1 using ionizing radiation-treated (6 Gy) extracts from NFFs transfected with ATM siRNA. d, ATM was immunoprecipitated from mock or ionizing radiation (6 Gy)-treated normal (C3ABR) and A-T (L3) cell lines. In vitro kinase assays were performed using recombinant hSSB1 as a substrate. e, Phosphorylation of hSSB1 (number denotes the position of threonine residue substituted with alanine) by immunoprecipitated ATM kinase. Vol 453 | 29 May 2008 | doi:10.1038/nature06883 677 Nature Publishing Group ©2008