Nucleic Acids Research, 1994, Vol. 22, No. 9 1527-1535
© 1994
MOLECULAR BIOLOGY |
The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae
1Department of Genetics, Sankt-Petersburg University Sankt-Petersburg, 199034, Russia 2Faculty of Parmaceutical Sciences, Okayama University Okayama 3Department of Molecular Immunology, Research Institute for Microbial Diseases Osaka University 3-1 Yamada-Oka, Suita, Osaka 565, Japan
*To whom correspondence should be addressed
Received March 3, 1994. Revised April 5, 1994. Accepted April 5, 1994.
Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.
+Present address: Department of Genetics, Sankt-Petersburg University, Sankt-Petersburg, Russia
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