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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cell Line, Tumor
  • Checkpoint Kinase 2 / genetics
  • Checkpoint Kinase 2 / metabolism
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • DNA Repair*
  • Histones / genetics
  • Histones / metabolism
  • Humans
  • Immunoblotting
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Microscopy, Fluorescence
  • Mitosis / genetics
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • RNA Interference
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Tumor Suppressor p53-Binding Protein 1

Substances

  • BUB2 protein, S cerevisiae
  • Bub1 protein, S cerevisiae
  • Cell Cycle Proteins
  • Histones
  • Intracellular Signaling Peptides and Proteins
  • Saccharomyces cerevisiae Proteins
  • TP53BP1 protein, human
  • Tumor Suppressor p53-Binding Protein 1
  • Checkpoint Kinase 2
  • BUB1 protein, human
  • Protein-Serine-Threonine Kinases
  • RAD53 protein, S cerevisiae