Mutational analysis of conserved sequence motifs in the budding yeast Cdc6protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (th e MCM complex) into prereplicative complexes at budding yeast origins of DN A replication. Cdc6p is a member of the AAA(+) superfamily of proteins, whi ch includes the prokaryotic and eukaryotic clamp loading proteins. These pr oteins share a number of conserved regions of homology and a common three-d imensional architecture. Two of the conserved sequence motifs are the Walke r A and B motifs that are involved in nucleotide metabolism and are essenti al for Cdc6p function in vivo. Here, we analyse mutants in the other conser ved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restr ictive temperature. In one such temperature-sensitive mutant, a highly cons erved asparagine residue in the sensor I motif was changed to alanine. Over expression 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 func tion. (C) 2001 Academic Press.