Abstract
The Cdc6 protein of budding yeast and its homologues in other species play an essential role in the initiation of DNA replication. A cDNA encoding a human homologue of Cdc6 (HsCdc6) has been cloned and expressed as a fusion protein in a soluble and functionally active form. The purified protein bound specifically to ATP and slowly hydrolyzed it, whereas HsCdc6 mutants containing amino acid substitutions in the Walker A or B motifs were defective. The mutant proteins retained the ability to bind HsOrc1 and HsCdc6 but displayed aberrant conformations in the presence of nucleotides. Microinjection of either mutant protein into human cells in G1 inhibited DNA replication, suggesting that ATP binding and hydrolysis by HsCdc6 are essential for DNA replication.
Publication types
-
Comparative Study
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, Non-P.H.S.
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Adenosine Triphosphate / metabolism
-
Animals
-
Baculoviridae / genetics
-
Binding Sites
-
Cell Cycle Proteins / genetics
-
Cell Cycle Proteins / isolation & purification
-
Cell Cycle Proteins / metabolism*
-
DNA Replication*
-
DNA Topoisomerases, Type II / chemistry
-
DNA Topoisomerases, Type II / genetics
-
DNA Topoisomerases, Type II / metabolism
-
DNA-Binding Proteins / genetics
-
DNA-Binding Proteins / metabolism
-
G1 Phase / genetics
-
HeLa Cells / metabolism
-
Humans
-
Hydrolysis
-
Kinetics
-
Microinjections
-
Molecular Sequence Data
-
Mutation
-
Nuclear Proteins / genetics
-
Nuclear Proteins / isolation & purification
-
Nuclear Proteins / metabolism*
-
Nucleotides / metabolism*
-
Origin Recognition Complex
-
Protein Conformation
-
Recombinant Proteins / genetics
-
Recombinant Proteins / isolation & purification
-
Recombinant Proteins / metabolism
-
S Phase / genetics
Substances
-
CDC6 protein, human
-
Cell Cycle Proteins
-
DNA-Binding Proteins
-
Nuclear Proteins
-
Nucleotides
-
Origin Recognition Complex
-
Recombinant Proteins
-
Adenosine Triphosphate
-
DNA Topoisomerases, Type II