Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
We report that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation. Supercoiling requires the initiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is prevented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, indicating that Cdc45, RPA, and DNA polymerase alpha bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these results for the location of replication start sites relative to the prereplication complex.
Walter
J
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. johannes_walter@hms.harvard.edu
Newport
J
J
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005057:Eukaryotic Cells; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018741:Replication Origin; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014981:Xenopus; D029867:Xenopus Proteins
C102209:CDC45 protein, S cerevisiae; D002352:Carrier Proteins; D002843:Chromatin; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D029867:Xenopus Proteins; C076713:P70 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004256:DNA Polymerase I; D004265:DNA Helicases