Repair Scaffolding Reaches New Heights at Blocked Replication Forks

Department of Biochemistry and Biophysics, University of California, San Francisco, 1450 3rd Street, San Francisco, CA 94158-9001, USA.
Molecular cell (Impact Factor: 14.02). 07/2010; 39(2):162-4. DOI: 10.1016/j.molcel.2010.07.007
Source: PubMed


Early studies of the DNA damage response focused on the relatively simple case of double-strand breaks. Lesions encountered during replication require a much more complicated series of events in which a cell must delay fork progression, repair or bypass the damage in question, and subsequently resume DNA synthesis—all without disrupting the delicate fork structure. This reorganization requires the recruitment of several repair and signaling complexes, in part facilitated by the recruitment of BRCT domain-containing proteins. BRCT domains recognize phosphorylated targets, in many cases mediated by the DNA damage-responsive kinase Mec1 (hATR). Several proteins important for resistance to replication stress, such as the yeast proteins Dpb11 (hTopBP1) and Rtt107, have several pairs of BRCT domains and thus may serve as scaffolds to coordinate multiple binding partners at sites of damage. In this issue, Ohouo et al. (2010) use mass spectrometry to expand the repertoire of proteins associated with these scaffolds in damage-treated cells. They show that Rtt107 associates with Dpb11, as well as the Slx4, Rtt101, and Smc5/Smc6 complexes.

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    ABSTRACT: Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT5-BRCT6) of Rtt107. The crystal structures of BRCT5-BRCT6 alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT5-BRCT6 and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.
    Journal of Biological Chemistry 01/2012; 287(12):9137-46. DOI:10.1074/jbc.M111.311860 · 4.57 Impact Factor


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