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Vol. 10, Issue 12, 3991-4003, December 1999
and
*Department of Molecular Genetics, Research Institute for Microbial
Diseases, Osaka University, Osaka 565-0871, Japan; and
We report here the isolation and functional analysis of the
rfc3+ gene of Schizosaccharomyces
pombe, which encodes the third subunit of replication factor C
(RFC3). Because the rfc3+ gene was essential
for growth, we isolated temperature-sensitive mutants. One of the
mutants, rfc3-1, showed aberrant mitosis with fragmented
or unevenly separated chromosomes at the restrictive temperature. In
this mutant protein, arginine 216 was replaced by tryptophan.
Pulsed-field gel electrophoresis suggested that rfc3-1
cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in
early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in
rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and
a significant reduction of the phosphorylated form of Chk1 in
rfc3-1 cells after treatments with MMS and gamma or UV
irradiation suggested that the checkpoint signal emitted by Rfc3 is
linked to the downstream checkpoint machinery via Rad17 and Chk1. From
these results, we conclude that rfc3+ is
required not only for DNA replication but also for replication and
damage checkpoint controls, probably functioning as a checkpoint sensor.
Department of Biology, Graduate School of Science, Osaka
City University, Osaka 558-8585, Japan
Corresponding author. E-mail address:
hnojima{at}biken.osaka-u.ac.jp.
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