Academic journal article Genetics

A SUMO-Like Domain Protein, Esc2, Is Required for Genome Integrity and Sister Chromatid Cohesion in Saccharomyces Cerevisiae

Academic journal article Genetics

A SUMO-Like Domain Protein, Esc2, Is Required for Genome Integrity and Sister Chromatid Cohesion in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Δ cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Δ rrm3Δ cells also have a severe growth defect and accumulate DNA damage in late S/G^sub 2^. In contrast, esc2Δ does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Δ cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Δ cells.

POST-TRANSLATIONAL modification, including phosphorylation, ubiquitination, and other types of covalent protein modification, is an important mechanism for rapidly altering protein stability, activity, or localization (Schwartz andHochstrasser 2003). The process/pathway of SUMOylation, which is mechanistically analogous toubiquitination, requiresadistinct group of SUMO-specific enzymes to covalently attach SUMO to its protein targets (Muller et al. 2001; Seeler and Dejean 2003; Johnson 2004). In contrast to ubiquitination, SUMOylation usually enhances the stability of protein targets or the formation of protein complexes and therefore plays a role in regulating multiple cellular processes, including subcellular localization, signal transduction, cell-cycle progression, and genome stability.

Saccharomyces cerevisiae ESC2 (Establishment silent chromatin 2) was first identified as genes necessary for silencing at mating-type locus that, when mutated or deleted, give rise to a partial defect in gene silencing (Dhillon and Kamakaka 2000; Cuperus and Shore 2002; Andrulis et al. 2004). Sequence analysis showed that Esc2 includes two tandem C-terminal SUMO-like domains and an N-terminal polar low-complexity domain, and its domain architecture is conserved in fission yeast (Rad60) and humans (NIP45) (Novatchkova et al. 2005). Although ESC2 is not essential for growth, Schizosaccharomyces pombe rad60 is essential for growth and rad60 mutants are hypersensitive to DNA damaging agents (Morishita et al. 2002; Boddy et al. 2003). The essential function of S. pombe Rad60 may be to regulate homologous recombination at stalled or collapsed DNA replication forks or to prevent cell cycle progression in cells with DNA damage (Miyabe et al. 2006; Raffa et al. 2006). Thus, since the functions known for Esc2 and Rad60 appear to be largely disparities, additional studies are needed to determine the functional similarities and/ or differences between S. cerevisiae Esc2 and S. pombe Rad60.

Tightly bound proteins or protein complexes and aberrant DNA structures can impede progression of the replication fork during S phase. Cells utilize several mechanisms, including DNA repair, DNA damage tolrerance, and DNA damage checkpoint pathways, to overcome such impediments and resume cell cycle progression (Cox et al. 2000; Barbour and Xiao 2003). Recent studies in yeast show that a Pif1-like 59 to 39 DNA helicase called Rrm3 facilitates restart of replication forks blocked by stable protein-DNA complexes (Ivessa et al. 2003). Cells that lack Rrm3 grow normally and are resistant to DNA damaging agents; however DNA replication is less processive due to frequent pausing, and an intra-S-phase/DNA damage checkpoint is activated in rrm3Δ cells (Torres et al. …

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