Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

Abstract

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00007-15.

ACKNOWLEDGMENTS

We are grateful to Vera Gorbunova (University of Rochester) for providing the NHEJ reporter system and Tina Singh from Grant Brown's laboratory (University of Toronto) for performing flow cytometry analysis. We thank the members of M. Babu's laboratory for technical assistance and Gabe Musso (Harvard Medical School, MA) and the anonymous reviewers of the manuscript for constructive suggestions, helpful discussions, and comments.

This research was supported by grants from the Natural Sciences and Engineering Research Council to A.G. and the Canadian Institutes of Health Research to M.B. (MOP-125952). M.B. holds a CIHR New Investigator award. M.J. and J.V. are recipients of a Saskatchewan Health Research Foundation Postdoctoral Fellowship.