Regulation of DNA replication machinery by Mrc1 in fission yeast

Genetics. 2006 Sep;174(1):155-65. doi: 10.1534/genetics.106.060053. Epub 2006 Jul 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins / genetics*
  • Cell Cycle Proteins / metabolism
  • Cell Cycle Proteins / physiology*
  • Checkpoint Kinase 1
  • Checkpoint Kinase 2
  • DNA Helicases / genetics
  • DNA Replication / drug effects
  • DNA Replication / physiology*
  • Hydroxyurea / pharmacology
  • Mutation
  • Nuclear Proteins / genetics
  • Origin Recognition Complex / genetics
  • Protein Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / physiology*
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Schizosaccharomyces pombe Proteins / physiology
  • Suppression, Genetic
  • Temperature

Substances

  • CDC45 protein, S pombe
  • Cell Cycle Proteins
  • MRC1 protein, S cerevisiae
  • Nuclear Proteins
  • Origin Recognition Complex
  • Saccharomyces cerevisiae Proteins
  • Schizosaccharomyces pombe Proteins
  • orc1 protein, S pombe
  • orc2 protein, S pombe
  • Protein Kinases
  • Checkpoint Kinase 2
  • Cds1 protein, S pombe
  • Checkpoint Kinase 1
  • Chk1 protein, S pombe
  • Protein-Serine-Threonine Kinases
  • DNA Helicases
  • Hydroxyurea