Roles of the CDK phosphorylation sites of yeast Cdc6 in chromatin binding and rereplication.
The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.
Honey
Sangeet
S
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222, USA.
Futcher
Bruce
B
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases; D004261:DNA Replication; D016195:G2 Phase / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D019913:Nuclear Localization Signals; D020411:Oligonucleotide Array Sequence Analysis; D051738:Origin Recognition Complex / Q000235:genetics; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D019913:Nuclear Localization Signals; C497215:ORC2 protein, S cerevisiae; C497221:ORC6 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; D015739:Nocodazole