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...

Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

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