Mol Cell Biol, August 1998, p. 4914-4923, Vol. 18, No. 8
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
andDepartment of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
Received 25 March 1998/Returned for modification 8 May 1998/Accepted 20 May 1998
Replication factor C (RF-C), an auxiliary factor for DNA
polymerases
and
, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds
to a primer terminus, and helps load proliferating cell nuclear antigen
onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of
this subunit in DNA metabolism, we isolated a thermosensitive mutation
(rfc2-1) in the RFC2 gene. It was shown that
mutant cells having the rfc2-1 mutation exhibit (i)
temperature-sensitive cell growth; (ii) defects in the integrity of
chromosomal DNA at restrictive temperatures; (iii) progression through
cell cycle without definitive terminal morphology and rapid loss of
cell viability at restrictive temperatures; (iv) sensitivity to
hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased
rate of spontaneous mitotic recombination and chromosome loss. These
phenotypes of the mutant suggest that the RFC2 gene product
is required not only for chromosomal DNA replication but also for a
cell cycle checkpoint. It was also shown that the rfc2-1
mutation is synthetically lethal with either the cdc44-1 or
rfc5-1 mutation and that the restrictive temperature of
rfc2-1 mutant cells can be lowered by combining
either with the cdc2-2 or pol2-11 mutation.
Finally, it was shown that the temperature-sensitive cell growth
phenotype and checkpoint defect of the rfc2-1
mutation can be suppressed by a multicopy plasmid containing the
RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and
RFC5 gene products in the RF-C complex and with both DNA
polymerases
and
during chromosomal DNA replication.
Present address: Department of Microbial Genetics, National
Institute of Genetics, 1-111, Yata, Mishima, Shizuoka 411-8540, Japan.
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