Regulation of DNA Replication Machinery by Mrc1 in Fission Yeast

Abstract

Faithful replication of chromosomes is crucial to genome integrity. In yeast, the ORC binds replication origins throughout the cell cycle. However, Cdc45 binds these before S-phase, and, during replication, it moves along the DNA with MCM helicase. When replication progression is inhibited, checkpoint regulation is believed to stabilize the replication fork; the detailed mechanism, however, remains unclear. To examine the relationship between replication initiation and elongation defects and the response to replication elongation block, we used fission yeast mutants of Orc1 and Cdc45-orp1-4 and sna41-928, respectively-at their respective semipermissive temperatures with regard to BrdU incorporation. Both orp1 and sna41 cells exhibited HU hypersensitivity in the absence of Chk1, a DNA damage checkpoint kinase, and were defective in full activation of Cds1, a replication checkpoint kinase, indicating that normal replication is required for Cds1 activation. Mrc1 is required to activate Cds1 and prevent the replication machinery from uncoupling from DNA synthesis. We observed that, while either the orp1 or the sna41 mutation partially suppressed HU sensitivity of cds1 cells, sna41 specifically suppressed that of mrc1 cells. Interestingly, sna41 alleviated the defect in recovery from HU arrest without increasing Cds1 activity. In addition to sna41, specific mutations of MCM suppressed the HU sensitivity of mrc1 cells. Thus, during elongation, Mrc1 may negatively regulate Cdc45 and MCM helicase to render stalled forks capable of resuming replication.

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

“…Furthermore, the mcb1 L254P mutant could partially suppress the HU sensitivity of the cds1⌬ cells (data not shown). This result suggests some defects of MCM function in mcb1 L254P mutant, because mutations in the MCM complex are known to suppress the HU sensitivity of the replication checkpoint mutants (62). All of these genetic results strongly indicate that the mcb1 L254P mutant has some defects in pre-RC formation, suggesting that Mcb1 has an important function in pre-RC formation.…”

Abundance of Prereplicative Complexes (Pre-RCs) Facilitates Recombinational Repair under Replication Stress in Fission Yeast

“…Yeast transformation was performed using the lithium acetate method (59). Centrifugal elutriation was performed as described previously (60).…”

Fission Yeast Swi1-Swi3 Complex Facilitates DNA Binding of Mrc1

“…Physical interaction of Tof1-Csm3 with Mrc1 was also reported (35). Also in fission yeast, Mrc1 has been suggested to negatively regulate Cdc45 and MCM helicase for recovery from replication block (12). However, little is known about the nature of the interaction between Mrc1 and Swi1-Swi3 (or these homologs) or about how this interaction could contribute to their actions at the fork.…”

“…The budding yeast Mrc1 protein and other orthologs of Mrc1 in Xenopus laevis and humans (known as Claspin) are also required for checkpoint activation at stalled forks, indicating that this aspect of Mrc1 function has been conserved throughout evolution (9 -11). Interestingly, recent genetic studies suggest checkpoint-independent functions of Mrc1 (12,13). However, the detailed mechanism remains unclear.…”