DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p

• Published in: The EMBO journal Vol. 19; no. 9; pp. 2082 - 2093
• Main Authors: Tercero, J.A, Labib, K, Diffley, J.F.X
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 02-05-2000; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Carrier Proteins - genetics; Carrier Proteins - metabolism; cdc45 gene; Cdc45p protein; cell cycle; Chromosomes, Fungal - drug effects; Chromosomes, Fungal - genetics; Deoxyribonucleic acid; DNA; DNA polymerase; DNA replication; DNA Replication - drug effects; DNA Replication - genetics; DNA, Fungal - biosynthesis; DNA, Fungal - genetics; DNA-Binding Proteins; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes, Essential - genetics; Hydroxyurea - pharmacology; Inactivation; initiation; interphase; mutants; Mutation - genetics; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; pre-replicative complex; replication origin; S Phase - drug effects; S-phase; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Simian virus 40; Temperature; Thermodynamics; Time Factors
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/19.9.2082

Cdc45p assembles at replication origins before initia tion and is required for origin firing in Saccharomyces cerevisiae. A heat‐inducible cdc45 degron mutant was constructed that promotes rapid degradation of Cdc45p at the restrictive temperature. Consistent with a role in initiation, loss of Cdc45p in G1 prevents all detectable DNA replication without preventing subsequent entry into mitosis. Loss of Cdc45p activity during S‐phase blocks S‐phase completion but not activation of replication checkpoints. Using density substitution, we show that after allowing replication fork establishment, Cdc45p inactivation prevents the subsequent progression of individual replication forks. This provides the first direct functional evidence that Cdc45p plays an essential role during elongation. Thus, like the large T antigen in SV40 replication, Cdc45p plays a central role in both initiation and elongation phases of chromosomal DNA replication.