HSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

Signal Transduction Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia.
Nucleic Acids Research (Impact Factor: 9.11). 11/2010; 39(5):1692-702. DOI: 10.1093/nar/gkq1098
Source: PubMed


hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

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    • "Unlike RPA however, hSSB1 is not required for normal S-phase progression (18). We and others have shown that hSSB1 is required for the recruitment of DNA repair factors to sites of DNA DSBs and stimulation of nucleases, such as Exonuclease 1 (Exo1) and the Mre11-Rad50-NBS1 (MRN) complex which resect the DNA to allow HR to proceed (20,21,22). "
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    ABSTRACT: Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.
    Full-text · Article · Apr 2014 · Nucleic Acids Research
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    • "Recently, SOSSB1 was reported to rapidly bind at the sites of DSBs and was required for the efficient recruitment of the MRN complex (Richard et al., 2011b). As SOSSB1 is one of the subunits of the SOSS1 complex, this result suggests that SOSS1 functions upstream of the MRN complex. "
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    ABSTRACT: The SOSS1 complex comprising SOSSA, SOSSB1, and SOSSC senses single-stranded DNA (ssDNA) and promotes repair of DNA double-strand breaks (DSBs). But how SOSS1 is assembled and recognizes ssDNA remains elusive. The crystal structure of the N-terminal half of SOSSA (SOSSAN) in complex with SOSSB1 and SOSSC showed that SOSSAN serves as a scaffold to bind both SOSSB1 and SOSSC for assembly of the SOSS1 complex. The structures of SOSSAN/B1 in complex with a 12 nt ssDNA and SOSSAN/B1/C in complex with a 35 nt ssDNA showed that SOSSB1 interacts with both SOSSAN and ssDNA via two distinct surfaces. Recognition of ssDNA with a length of up to nine nucleotides is mediated solely by SOSSB1, whereas neither SOSSC nor SOSSAN are critical for ssDNA binding. These results reveal the structural basis of SOSS1 assembly and provide a framework for further study of the mechanism governing longer ssDNA recognition by the SOSS1 complex during DSB repair.
    Full-text · Article · Mar 2014 · Cell Reports
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    • "The MRN components are thus the only sensors that may be used to observe DSBs, despite their foci formation being delayed [50]. However, the human ssDNA binding protein 1 (hSSB1), binding ssDNA overhangs of IR-induced DSBs, should be considered as a new DSB sensor as it localizes to DSB with the same kinetics than MRN, and is essential for ATM signaling and MRN recruitment [51] [52]. Moreover, due to its role in later HR steps, hSSB1 remains associated longer than MRN to DSBs, and may thus provide a more stable DSB biomarker. "
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    ABSTRACT: The occurrence of DNA double-strand breaks (DSBs) induced by ionizing radiation has been extensively studied by biochemical or cell imaging techniques. Cell imaging development relies on technical advances as well as our knowledge of the cell DNA damage response (DDR) process. The DDR involves a complex network of proteins that initiate and coordinate DNA damage signaling and repair activities. As some DDR proteins assemble at DSBs in an established spatio-temporal pattern, visible nuclear foci are produced. In addition, post-translational modifications are important for the signaling and the recruitment of specific partners at damaged chromatin foci. We briefly review here the most widely used methods to study DSBs. We also discuss the development of indirect methods, using reporter expression or intra-nuclear antibodies, to follow the production of DSBs in real time and in living cells.
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