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

Alexandra M Locovei,Ling Yin,Gennaro D'Urso

doi:10.1186/1747-1028-5-20

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.

Highlights

Of DNA replication complexes begins in early G1 following degradation of cyclin-dependent kinases at the conclusion of mitosis [1]
Replication initiation mutants have unique checkpoint requirements Previously, we showed that several mutants defective in DNA replication initiation are sensitive to the loss of chk1+, but not cds1+ [26], see Additional file 1, Table S1
In this report we have shown that mutants defective for DNA replication initiation require Chk1, but not Cds1 to maintain cell viability

Summary

Introduction

Of DNA replication complexes begins in early G1 following degradation of cyclin-dependent kinases at the conclusion of mitosis [1]. The dramatic drop in CDK activity following anaphase promotes the binding of Mcm to origin DNA to form pre-replicative complexes or pre-RCs. Formation of the pre-RC requires both Cdc18/Cdc and Cdt proteins [2,3,4]. Once the pre-RC is assembled, activation of CDK (cyclin dependent kinase) and DDK (Dbf4-dependent kinase) at the beginning of S phase leads to the binding of additional replication proteins to origin DNA including DNA polymerase epsilon, Sld, Sld, Cdc, and GINS forming the pre-initiation complex or pre-IC [5,6]. DDK and CDK are two serine kinases required for the onset of S phase They target components of the pre-RC and pre-IC, respectively and are essential for initiation of DNA replication. Mcm proteins contain ATP binding sites but ATP hydrolysis is only required for replication elongation and not for chromatin binding [15]

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Results

Conclusion