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Mol Cell Biol. 2015 Jul;35(14):2448-63. doi: 10.1128/MCB.00007-15. Epub 2015 May 11.

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

Author information

1
Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan, Canada Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario, Canada.
2
Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan, Canada.
3
Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario, Canada.
4
Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan, Canada Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
5
Cancer Research Unit, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada.
6
Department of Medicine, Regina Qu'Appelle Health Region, Regina, Saskatchewan, Canada.
7
Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
8
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
9
Department of Biology and Ottawa Institute of Systems Biology, Carleton University, Ottawa, Ontario, Canada ashkan_golshani@carleton.ca mohan.babu@uregina.ca.
10
Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina, Saskatchewan, Canada ashkan_golshani@carleton.ca mohan.babu@uregina.ca.

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.

PMID:
25963654
PMCID:
PMC4475915
DOI:
10.1128/MCB.00007-15
[Indexed for MEDLINE]
Free PMC Article

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