Nse1, Nse2, and a Novel Subunit of the Smc5-Smc6 Complex, Nse3, Play a Crucial Role in Meiosis

Abstract

The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1؉3) holds replicated sister chromatids together until mitosis, condensin (Smc2؉4) acts in chromosome condensation, and Smc5؉6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5؉6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5؉6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5؉6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5؉6 complex is important for replication fork stability. INTRODUCTIONBoth endogenous and exogenous agents constantly threaten genomic integrity. The DNA double-strand break (DSB) is a potentially life-threatening lesion for a cell and can occur spontaneously during growth, as part of a programmed event such as meiosis or as the result of exposure to environmental genotoxic agents. Multiple mechanisms exist to repair DSBs, including nonhomologous end joining and homologous recombination (HR) (Paques and Haber, 1999;Symington, 2002). The HR pathway serves to maintain all original genetic information during DSB repair. Many components of that pathway have been identified and characterized (Paques and Haber, 1999;Symington, 2002). HR involves multiple steps. The DSB is first resected to generate a recombinogenic 3Ј overhang that invades a homologous sequence (e.g., sister chromatid), forming a displacement or D-loop. The invasion step is catalyzed by a number of proteins including the Escherichia coli RecA homologue, Rad51. D-loop formation primes repair synthesis, which is followed by resolution of the recombined duplexes and ligation, producing intact duplex DNA molecules (Paques and Haber, 1999;Symington, 2002). The resolution of recombined DNA duplexes can yield products in which there has been reciprocal exchange of flanking markers (crossover) or not. During mitotic growth gene conversion is infrequently accompanied by crossover, whereas during meiosis, crossover recombination is much more prevalent (Paques and Haber, 1999).Efficient DNA repair requires modulation of both local and higher order chromatin structure. Interestingly, the structural maintenance of chromosomes (SMC) proteins have recently been recognized as important players in DNA repair (Hirano, 2002;Jessberger...

During Replication Stress, Non-Smc Element 5 (Nse5) Is Required for Smc5/6 Protein Complex Functionality at Stalled Forks

“…During Replication Stress, Defects Are Observed in nse5-ts1 Cells-Previous reports have demonstrated that hypomorphic alleles of Smc5/6 components are very sensitive to HU, indicating that the complex likely has a fundamental role when forks stall (11)(12)(13)(14)(15)(16)(17)(18). Drop assays showed that nse5-ts1 (but not nse5-ts2) cells are sensitive to HU (Fig.…”

“…Moreover, to date, the visualization of recombination intermediates by two-dimensional gel electrophoresis in Smc5/ 6-deficient cells has been limited to either cells that have been treated with MMS or cells in which replication forks have collapsed (9,12). Nonetheless, many Smc5/6 hypomorphic alleles are very sensitive to the drug HU, which suggests that the complex also has a fundamental role when forks stall prior to collapse (11)(12)(13)(14)(15)(16)(17)(18).…”

Sumoylation: A new wrestler in the DNA repair ring

“…An interacting protein, Spr18, also found initially in Schizosaccharomyces pombe, turn out to be the heterodimeric partner, Smc5p (17). Since then, the Non-Smc Elements in the complex has grown to four in Schizosaccharomyces pombe (Nse1-4) (8,(18)(19)(20) and six in S. cerevisiae (Nse1-6) (1,21,22). The Smc5-6 complex is involved in damage repair because all mutants identified to date exhibit sensitivities to a variety of DNA damaging agents.…”

“…However, the complex is also thought to carry out an essential function, and presently it is unclear to what extent the DNA repair and essential functions overlap. Temperature-sensitive mutants of the Schizosaccharomyces pombe rad18 (SMC6) gene exhibit a nonspecific terminal phenotype where cells are able to go through a few rounds of division before arresting growth (19). As mutant cells divide, the appearance of a ''cut'' phenotype, where incompletely separated nuclei are cut by the formation of a new septum, suggests a requirement for the accurate transmission of chromosomes.…”

When MAGE meets RING: insights into biological functions of MAGE proteins

“…Exciting progress in this field was achieved when several groups' data are pointing to RING domain proteins as binding partners for both type I and type II MAGEs (Pebernard et al, 2004;Rual et al, 2005;Sergeant et al, 2005;Yang et al, 2007;Taylor et al, 2008). For example, the interspersed repeat domain of NRAGE/MAGE-D1 (herein referred to as MAGE-D1 for simplicity) was identified to bind RING domain protein XIAP and the avian IAP homolog ITA, members of the inhibitor of apoptosis (IAP) family (Jordan et al, 2001).…”