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 plasmidbased 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.T he repair of DNA double-strand breaks (DSBs) is an essential process required for the preservation of genome integrity and the normal functioning of the cell (1). These cytotoxic lesions are repaired by major DSB repair pathways, including the homologous recombination (HR) (2) and nonhomologous end-joining (NHEJ) systems (1). While the former is the prevalent pathway in the unicellular budding yeast Saccharomyces cerevisiae (3), the latter is more prevalent in mammalian cells, especially those that are quiescent (4), and can repair DNA lesions even if there is no homologous strand (5). Notably, the impairment of NHEJ in mammalian cells is frequently linked to genomic instability, cancer, and lymphoid V(D)J (i.e., variable, diversity, and joining gene segments) recombination defects. Therefore, a detailed molecular understanding of this pathway would provide critical insight into the genetic risk factors related to carcinogenesis or immunological disorders (6).As in mammalian cells, the core components of the classical NHEJ pathway in S. cerevisiae depends on three major complexes, YKu (Ku), MRX, and DNL4, which are rapidly recruited to DSBs (7). Initially, the yeast Ku heterodimer (Ku70/80) binds to each end of a DSB, serving as an anchor for protein complexes involved in securing and annealing the break, also suppressing the competing HR pathway (8). After this, the DSB processing complex MRX (Mre11-Rad50-Xrs2), which acts at an early stage of both the NHEJ and HR repair pathways (9), spans the lesions so that the DNA ligase complex, DNL4 (i.e., Dnl4-Lif1-Nej1), can rejoin the DSB ends (10).While the actions of these core protein complexes in y...

Consider the workhorse: Nonhomologous end-joining in budding yeast

“…Indeed, several NHEJ factors and processes were first characterized in S. cerevisiae , underscoring the organism’s significance in the field of DSB repair. Large scale screens in yeast continue to identify new genes that influence NHEJ (Jessulat et al 2015; McKinney et al 2013). Additionally, because aberrant NHEJ is a major source of chromosomal disruption in budding yeast (Yu and Gabriel 2004), like mammalian cells (Lieber et al 2010), S. cerevisiae provides an appealing model to study the implications of NHEJ defects in human genome instability.…”

Identification and Investigation of Novel DNA Damage Repair Genes Via Network Analysis

“…Despite these qualities, NHEJ is an essential process across all eukaryotes, capable of repairing an array of broken ends and doing so independently of a repair template 13 . The core machinery involved in classical NHEJ (c-NHEJ) is highly conserved between human and yeast cells, comprising the Ku70/80 heterodimer, the MRX complex, and ligase proteins such as LIG4 in human cells or the complex DNL4 in yeast 8,14 . KU70 and KU80 (Yku70 and Yku80 in yeast) act as first responders, binding to the exposed DNA ends and preventing HR machinery from initiating end resection.…”

“…KU70 and KU80 (Yku70 and Yku80 in yeast) act as first responders, binding to the exposed DNA ends and preventing HR machinery from initiating end resection. Ku70/80 then serves as a docking site, recruiting nuclease complex MRX to process the ends, as well as polymerases (Pol4 in yeast) and ligases or ligase complexes (such as DNL4, comprised of Dnl4, Lif1, and Nej1) to initiate re-ligation 13,14 . 14 There exist several NHEJ subclasses of repair aside from c-NHEJ.…”

“…Candidate genes GAL7, YHI9, and YMR130W were identified by first compiling a nonredundant subset of genes with known NHEJ involvement and a set of nonessential DNA damage related genes previously identified as having significant (mutant repair efficiency<50%) impact on repair of DSBs 14 . The set was then used to generate a putative 34 interaction network through GeneMANIA software 58 .…”

“…Applying the 'guilt-byassociation' principle to network biology, we hypothesized that by employing simple analysis of an NHEJ-related network, we might discover novel genes involved in DSB repair. Emphasis was placed on protein-protein interactions, as these have shown to be reliable indicators of function in the past 14 .…”