Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.
The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.
Schepers
A
A
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, EN6 3LD, UK.
Diffley
J F
JF
D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D002843:Chromatin / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005799:Genes, Dominant / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D064168:Minichromosome Maintenance Complex Component 7; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7