Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions
- PDF / 486,552 Bytes
- 14 Pages / 595 x 785 pts Page_size
- 32 Downloads / 110 Views
O R I GI N A L P A P E R
W. Beckwith á M. A. McAlear
Allele-speci®c interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions
Received: 7 April 2000 / Accepted: 20 July 2000 / Published online: 31 August 2000 Ó Springer-Verlag 2000
Abstract Replication factor C (RFC) is an essential, multi-subunit ATPase that functions in DNA replication, DNA repair, and DNA metabolism-related checkpoints. In order to investigate how the individual RFC subunits contribute to these functions in vivo, we undertook a genetic analysis of RFC genes from budding yeast. We isolated and characterized mutations in the RFC5 gene that could suppress the cold-sensitive phenotype of rfc1-1 mutants. Analysis of the RFC5 suppressors revealed that they could not suppress the elongated telomere phenotype, the sensitivity to DNA damaging agents, or the mutator phenotype of rfc1-1 mutants. Unlike the checkpoint-defective rfc5-1 mutation, the RFC5 suppressor mutations did not interfere with the methylmethane sulfonate- or hydroxyurea-induced phosphorylation of Rad53p. The Rfc5p suppressor substitutions mapped to amino acid positions in the conserved RFC box motifs IV±VII. Comparisons of the structures of related RFC box-containing proteins suggest that these RFC motifs may function to coordinate interactions between neighboring subunits of multisubunit ATPases. Key words Replication factor C á Saccharomyces cerevisiae á RFC1 á RFC5 á DNA replication
Introduction Numerous studies have revealed that the underlying molecular mechanisms of many DNA replication and DNA repair processes are conserved between prokary-
Communicated by T. D. Fox W. Beckwith á M. A. McAlear (&) Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT, 06459-0175, USA E-mail: [email protected] Fax: +1-860-685-2141
otes and eukaryotes. In many cases, the polymerization of nucleotides is carried out by multi-protein complexes containing DNA polymerases and their accessory proteins (reviewed in Stillman 1994). Among the important accessory proteins that promote DNA polymerase activity are the sliding DNA clamps and the clamp loaders. Sliding DNA clamps are ring-shaped proteins that can encircle DNA and tether DNA polymerases to the DNA templates (Stillman 1994). Detailed analysis has revealed an impressive degree of conservation within the structure of the clamps from various systems, including the eukaryotic proliferating cell nuclear antigen (PCNA), the b subunit of DNA polymerase III from E. coli, and the gp45 protein from phage T4 (Kong et al. 1992; Krishna et al. 1994). In order for these proteins to function as sliding DNA clamps, they must ®rst be wrapped around the DNA template by the clamp loaders. The clamp loader proteins [e.g. Replication Factor C (RFC) in eukaryotes, the c complex in E. coli and the gp44/62 complex from T4] are multi-subunit DNA-binding proteins that load their respective clamp proteins onto DNA in an ATP-dependent reaction (Yao et al. 1996). Studies on the c complex i
Data Loading...
