Premature structural changes at replication origins in a yeast minichromosome maintenance (MCM) mutant.
The Cdc7p protein kinase in the budding yeast Saccharomyces cerevisiae is thought to help trigger DNA replication by modifying one or more of the factors that assemble at replication origins (ARSs). To investigate events catalyzed by Cdc7p, we compared the structure of replication origins in cells containing conditional mutations in Cdc7p and Cdc8p, a thymidylate kinase that is required for DNA synthesis. High resolution genomic footprinting indicated that the presumptive lagging strand template in ARS1 became highly sensitive to KMnO(4) modification after the CDC7 execution point. These results suggested that Cdc7p triggers DNA unwinding. The transition from late G(1) phase to the CDC7 execution point and from the CDC7 to the CDC8 execution points was accompanied by small but ARS-dependent changes in DNA topology. These results suggested that DNA unwinding before the CDC8 execution point either is highly localized or that the torsional stress associated with initial DNA unwinding is minimized by compensatory protein-DNA structural changes. The ARS DNA structural attributes evident in cells blocked at the CDC8 execution point were also evident in alpha-factor-blocked, G(1) phase cells containing the CDC7 bypass mutant mcm5/cdc46-bob1. This result strongly suggests that the structural changes during the transition from the CDC7 to CDC8 execution points depend on the Cdc7p protein kinase and involve alteration of the minichromosome maintenance protein complex.
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Departments of Microbiology and Molecular Genetics and Pathology and the Markey Center for Molecular Genetics, University of Vermont School of Medicine, Burlington, Vermont 05405, USA.
Ding
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Heintz
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Pederson
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DS
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018983:DNA Footprinting; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D011196:Potassium Permanganate / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; D011196:Potassium Permanganate; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D008836:Micrococcal Nuclease