A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe

Alexandra M. Locovei, Ling Yin, Gennaro D'urso

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation.

Original languageEnglish
Article number20
JournalCell Division
Volume5
DOIs
StatePublished - Aug 27 2010

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Schizosaccharomyces
DNA Replication
DNA
Cells
DNA Damage
Cell Cycle
S Phase Cell Cycle Checkpoints
S Phase
Phenotype
Elongation
Cell Survival
Yeast
Mutation
Temperature
Phosphotransferases
G2 Phase
Chemical activation
DNA-Directed DNA Polymerase
DNA Repair

ASJC Scopus subject areas

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Cite this

A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe. / Locovei, Alexandra M.; Yin, Ling; D'urso, Gennaro.

In: Cell Division, Vol. 5, 20, 27.08.2010.

Research output: Contribution to journalArticle

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