| dc.description.abstract | The repair of DNA interstrand crosslinks (ICLs) involves the concerted action of multiple DNA repair pathways. In eukaryotes, ICLs are sensed and repaired by replication-coupled or replication-independent mechanisms in which PSO2/SNM1 is essential for unhooking an ICL. Previous work has shown that ICL-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA (mtDNA), and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mtDNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. In the first part of this study, we provide direct evidence that the S. cerevisiae nuclear-encoded Pso2 is a dual-localized ICL repair factor: it is imported into mitochondria and the nucleus, and its abundance increases in the mitochondria following treatment with DNA damaging agents. We reveal that Pso2 contains one mitochondrial targeting sequence (MTS) and two nuclear localization signals (NLS1 and NLS2) in its N-terminus, although NLS1 resides within the MTS. While MTS is necessary to guide the passage of Pso2 into the mitochondrial matrix, either NLS1 or NLS2 is sufficient for its import into the nucleus, implying that the two NLS motifs are functionally redundant. Ablation of MTS abrogated mitochondrial import of Pso2, and concomitant enrichment in the nucleus. Strikingly, mutations that render both NLS1 and NLS2 inactive, blocked nuclear import of Pso2; at the same time, increased its mitochondrial localization, consistent with the notion that the relationship between MTS and NLS motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 to two genome-bearing organelles is discussed
In the second part of this study, we combined a range of methods and approaches to determine the cell-intrinsic and extrinsic factors that regulate the intracellular distribution and function of Pso2 in ICL repair. Indeed, these approaches demonstrated that Pso2 is modified by ubiquitination and phosphorylation (but not SUMOylation) under normal growth conditions. Notably, we found that SUMOylation of Pso2 and its subsequent import into the mitochondria was induced by methyl methanesulfonate (MMS), but not cisplatin. We also demonstrate that the levels of MMS-induced Pso2 SUMOylation remain relatively constant throughout the synchronized cell cycle. Mutation of lysine residues (K97 and K575) within the SUMO consensus motifs in Pso2 abolished its import into the mitochondria. We further show that SIZ1 and SIZ2 SUMO E3 ligases act redundantly to catalyze SUMOylation of Pso2, while the former plays a dominant role in this process. Quite unexpectedly, we found that PSO1 (but not PSO2) was essential for attenuation of MMS-induced cell death; this implies that they function in non-overlapping ICL repair pathways or the former is epistatic to the latter. The findings reveal novel mechanistic insights into the regulation and intracellular distribution of Pso2 and shed new light on its function in DNA repair. | en_US |
