Prp20, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, RCC1, interacts with double-stranded DNA through a multi-component complex containing GTP-binding proteins.
Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains. Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In vitro reconstitution assays using E. coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da. At least three of the members of this Prp20 complex will bind GTP in vitro. Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle. This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.
Lee
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Department of Biology, McGill University, Montréal, Québec, Canada.
Tam
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Belhumeur
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DiPaolo
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Clark
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D018797:Cell Cycle Proteins; D004247:DNA / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D020662:Guanine Nucleotide Exchange Factors; D020559:Monomeric GTP-Binding Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C080324:GSP1 protein, S cerevisiae; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C067050:SRM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D019204:GTP-Binding Proteins; C080325:GSP2 protein, S cerevisiae; D020559:Monomeric GTP-Binding Proteins