Linked Life Data

10075735

Source:http://linkedlifedata.com/resource/pubmed/id/10075735

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
12
pubmed:dateCreated
1999-4-15
pubmed:abstractText
Saccharomyces cerevisiae Cdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
274
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8291-8
pubmed:dateRevised
2008-10-30
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association.
pubmed:affiliation
Department of Pediatrics, University of Southern California, Los Angeles, California 90027, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't