Abstract
Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.
Highlights
Condensin is a hetero-pentameric protein complex in eukaryotes that consists of two structural maintenance of chromosomes (SMC) subunits and three regulatory non-SMC subunits [1,2,3,4,5,6]
We show that condensin antagonizes replication protein A (RPA) [22 –25] activity by removing it from DNA in vitro and in vivo, which suggests that the DNA reannealing activity of condensin may facilitate the removal of proteins from chromosomes after DNA repair or prior to chromosome segregation
After examining more mutants involved in DNA and RNA metabolism by tetrad dissection, we found that one mutant, ssb1-418, showed a striking synthetic rescue of the cut14-Y1 ts phenotype at 338C, and the HU and UV sensitivities at 268C. ssb1-418 is a mutant of Ssb1, the largest subunit of heterotrimeric RPA
Summary
Condensin is a hetero-pentameric protein complex in eukaryotes that consists of two structural maintenance of chromosomes (SMC) subunits and three regulatory non-SMC subunits [1,2,3,4,5,6]. The diverse roles of condensin in chromosome dynamics, including mitotic chromosome condensation and segregation, DNA metabolism and development, are well documented [15,16,17,18,19], but the molecular mechanism of how it functions is not well understood. We show that condensin antagonizes replication protein A (RPA) [22 –25] activity by removing it from DNA in vitro and in vivo, which suggests that the DNA reannealing activity of condensin may facilitate the removal of proteins from chromosomes after DNA repair or prior to chromosome segregation
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