Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex
ABSTRACT The structural maintenance of chromosomes (SMC) family of proteins play essential roles in genomic stability. SMC heterodimers are required for sister-chromatid cohesion (Cohesin: Smc1 & Smc3), chromatin condensation (Condensin: Smc2 & Smc4), and DNA repair (Smc5 & Smc6). The SMC heterodimers do not function alone and must associate with essential non-SMC subunits. To gain further insight into the essential and DNA repair roles of the Smc5-6 complex, we have purified fission yeast Smc5 and identified by mass spectrometry the co-precipitating proteins, Nse1 and Nse2. We show that both Nse1 and Nse2 interact with Smc5 in vivo, as part of the Smc5-6 complex. Nse1 and Nse2 are essential proteins and conserved from yeast to man. Loss of Nse1 and Nse2 function leads to strikingly similar terminal phenotypes to those observed for Smc5-6 inactivation. In addition, cells expressing hypomorphic alleles of Nse1 and Nse2 are, like Smc5-6 mutants, hypersensitive to DNA damage. Epistasis analysis suggests that like Smc5-6, Nse1, and Nse2 function together with Rhp51 in the homologous recombination repair of DNA double strand breaks. The results of this study strongly suggest that Nse1 and Nse2 are novel non-SMC subunits of the fission yeast Smc5-6 DNA repair complex.
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ABSTRACT: Genomic integrity is an absolute requirement for cell survival. Programmed events such as genome rearrangements and DNA replication can cause lesions in the DNA, as can exogenous agents such as radiation and chemicals. One of the most austere types of lesion is DNA double strand breaks (DBSs). In Saccharomyces cerevisiae, budding yeast, they are preferentially repaired by the homologous recombination (HR) pathway using a homologous DNA sequence as template. The Structural maintenance of chromosomes (SMC) family proteins are essential for cell viability and have functions in chromosome condensation, segregation and in DNA repair by HR. The cohesin complex is important for cohesion and correct segregation of sister chromatids. The Smc5/6 complex functions late in the HR process and it has another function, not yet entirely elucidated, that makes the complex essential. We have investigated the chromosomal localization of the Smc5/6 complex and found that the complex associates with specific sites along the chromosome arms in a chromosome length-dependent manner. This association is dependent on the cohesin loading protein Scc2. The complex also localizes to chromosomal regions surrounding a DNA DSB in the G2/M phase. Localization to DSBs is dependent on the damage-sensing HR protein Mre11, but not on Scc2. Smc6 mutants exhibit a delay in chromosome segregation and a closer investigation suggests that this delay is caused by persisting replication forks. The length-dependent distribution of the Smc5/6 complex on chromosomes was found to reflect a function of the complex that is independent of its function in HR. A possible explanation for this length-dependency is the accumulation of replication-induced topological structures on longer chromosomes due to their inability to swivel off the torsional stress. A circular short chromosome is therefore expected to generate more unresolved topological structures than a linear version of the same chromosome. Smc5/6 complex components showed an increase in binding regions on a circular chromosome compared to the linear version. Deletion of Top1, a protein required for release of replication-induced torsional tension in DNA, also shows a similar chromosome length-specific phenotype as the Smc5/6 complex components, indicating that topology is the inherent cause of the Smc5/6 complex association with chromosomes.
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ABSTRACT: ABSTRACT The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of two winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast two-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Amongst these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.Cell cycle (Georgetown, Tex.) 03/2015; 14(6):920-930. DOI:10.1080/15384101.2014.1000112
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ABSTRACT: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes.Genome Medicine 01/2014; 6(9):68. DOI:10.1186/s13073-014-0068-4