We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro.
- Authors
Kawasaki, Yasuo; Kim, Hee-Dai; Kojima, Akihiro; Seki, Takashi; Sugino, Akio
- 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.
- Publication
Genes to cells : devoted to molecular & cellular mechanisms, 2006, Vol 11, Issue 7, p745
- ISSN
1356-9597
- Publication type
Journal Article
- DOI
10.1111/j.1365-2443.2006.00975.x