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Kawasaki Y, et al.  (2006) Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro. Genes Cells 11(7):745-56

Abstract: The assembly of the prereplicative complex (pre-RC) at the origin of replication in eukaryotes is a highly regulated and highly conserved process that plays a critical role in preventing multiple rounds of DNA replication per cell division cycle. This study analyzes the molecular dynamics of the assembly of Saccharomyces cerevisiae pre-RC in vitro using ARS1 plasmid DNA and yeast whole cell extracts. In addition, pre-RC assembly was reconstituted in vitro using ARS1 DNA and purified origin-recognition complex (ORC), Cdc6p and Cdt1p-Mcm2-7p. The results reveal sequential recruitment of ORC, Cdc6p, Cdt1p and Mcm2-7p on to ARS1 DNA. When Mcm2-7p is maximally loaded, Cdc6p and Cdt1p are released, suggesting that these two proteins are co-ordinately regulated during pre-RC assembly. In extracts from sid2-21 mutant cells that are deficient in CDT1, ORC and Cdc6p bind to ARS1 but Cdt1p and Mcm2-7p do not. However, Mcm2-7p does bind in the presence of exogenous Cdt1p or Cdt1p-Mcm2-7p complex. Cdt1p-Mcm2-7p complex, which was purified from G1-, early S or G2/M-arrested cells, exhibits structure-specific DNA binding, interacting only with bubble- or Y-shape-DNA, but the biological significance of this observation is not yet known.

Status: Published Type: Journal Article PubMed ID: 16824194

Topics addressed in this paper

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Topics Genes linked to topics (#1 - 10 )
ABF1 ARS1 CDC6 MCM2 MCM3 MCM4 MCM5 MCM6 MCM7 ORC1
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Topics Genes linked to topics (#11 - 16 )
ORC2 ORC3 ORC4 ORC5 ORC6 TAH11
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