PRP20, THE SACCHAROMYCES-CEREVISIAE HOMOLOG OF THE REGULATOR OF CHROMOSOME CONDENSATION, RCC1, INTERACTS WITH DOUBLE-STRANDED DNA THROUGH AMULTICOMPONENT COMPLEX CONTAINING GTP-BINDING PROTEINS

Prp20, a homolog to the mammalian negative regulator of chromosome con densation, RCC1, is retained on double-stranded (ds) DNA-cellulose whe n extracts are prepared from asynchronously growing wild-type yeast st rains. Conversely, neither Prp20 from ts mutant cell extracts nor wt y east Prp20 produced in Escherichia coli, bind to dsDNA-cellulose. In v itro reconstitution assays using E. coli-expressed Prp20 and inactivat ed ts mutant extracts of prp20-1 reveal that the Prp20 protein require s the assistance of other proteins in the cell extract to promote its binding to dsDNA. Immunoprecipitations and sizing-column-chromatograph y 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 t he 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 dsDN A binding during the S phase of the cell cycle does not affect the pro per organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.