Different requirements for the association of ATR-ATRIP and 9-1-1 to the stalled replication forks.
DNA replication checkpoint, a surveillance mechanism for S-phase progression, plays a crucial role for the maintenance of genome integrity. A variety of factors have been characterized to be involved in the checkpoint signal transduction. Rpa, a single strand DNA binding protein, was found to be responsible for forming a structure that is recognized by checkpoint sensors and then emits the initial signal for the activation of DNA damage checkpoint. Here we use a mutant of rpa1 gene, rfa1-t11, that has defects in recruiting checkpoint sensor proteins to the site of double strand break, to examine the mutant's effects on the activation of DNA replication checkpoint. We found that the mutant cells activated DNA replication checkpoint normally and showed no defects in recruiting ATR-ATRIP, a major sensor complex that is essential for DNA replication/damage checkpoint, to the site of stalled forks. In contrast, the mutant was defective in recruiting 9-1-1 complex, another sensor complex that functions in DNA damage checkpoint signal transduction, to the stalled forks. Moreover we found that sensitivity for HU obviously appeared in rfa1-t11 mutant when Mrc1 was deleted, while deletion of Rad9, an adaptor specific for damage checkpoint, had subtle effect. These data strongly suggest that rfa1-t11 mutant was mainly defective for activating DNA damage checkpoint and molecular requirement for the recruitment of ATR-ATRIP and 9-1-1 to the stressed forks may be different.
Kanoh
Yutaka
Y
Laboratory of Genome Structure and Function, Division for Gene Research, Center for Biological Resources and Informatics, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama City, Kanagawa, Japan.
Tamai
Katsuyuki
K
Shirahige
Katsuhiko
K
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D046911:Macromolecular Substances; D009154:Mutation; D016133:Polymerase Chain Reaction; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors