Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases.
Eukaryotic cells use multiple replication origins to replicate their large genomes. Some origins fire early during S phase whereas others fire late. In Saccharomyces cerevisiae, initiator sequences (ARSs) are bound by the origin recognition complex (ORC). Cdc6p synthesized at the end of mitosis joins ORC and facilitates recruitment of Mcm proteins, which renders origins competent to fire. However, origins fire only upon the subsequent activation of S phase cyclin-dependent kinases (S-CDKs) and Dbf4/Cdc7 at the G1/S boundary. We have used a chromatin immunoprecipitation assay to measure the association with ARS sequences of DNA primase and the single-stranded DNA binding replication protein A (RPA) when fork movement is inhibited by hydroxyurea (HU). RPA's association with origins requires S-CDKs, Dbf4/Cdc7 kinase and an Mcm protein. The recruitment of DNA primase depends on RPA. Furthermore, early- and late-firing origins differ not in the timing of their recruitment of an Mcm protein, but in the timing of RPA's recruitment. RPA is recruited to early but not to late origins in HU. We also show that Rad53 kinase is required to prevent RPA association with a late origin in HU. Our data suggest that the origin unwinding accompanied by RPA association is a key step, regulated by S-CDKs, Dbf4/Cdc7 and Rad53p. Thus, in the presence of active S-CDKs and Dbf4/Cdc7, Mcms may open origins and thereby facilitate the loading of RPA.
Tanaka
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T
Research Institute of Molecular Pathology, A-1030 Vienna, Austria.
Nasmyth
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K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D015825:Chromosomes, Fungal / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004265:DNA Helicases; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D006918:Hydroxyurea / Q000494:pharmacology; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D018741:Replication Origin; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D009687:Nuclear Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; C067022:RAD53 protein, S cerevisiae; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7; D006918:Hydroxyurea