Analyses of Saccharomyces cerevisiae Cdc7 kinase point mutants: dominant-negative inhibition of DNA replication on overexpression of kinase-negative Cdc7 proteins.
Saccharomyces cerevisiae Cdc7 kinase is required for initiation of S phase, and its kinase activity, which is positively regulated by Dbf4 protein, reaches maximum at the G1/S boundary. In this study, we constructed Cdc7 point mutants (T281E, T281A, D182N, D163N, and T167E) and examined the effect of each mutant on growth. All the mutants lost the ability to complement temperature-sensitive growth of cdc7(ts) mutants at a low protein level, whereas T281A (putative target of phosphorylation) and T167E (residue involved in substrate recognition) restored the growth of cdc7(ts) when overproduced to a high level. Three putative kinase-negative mutants (T281E, D182N, and D163N) inhibited growth when overexpressed in a wild-type strain. Analyses of DNA content and morphology revealed that most cells were arrested as dumbbells with 1C DNA, indicative of a block in the G1 to S transition. This growth inhibition was suppressed by co-overexpression of the wild-type Cdc7 or Dbf4 protein. Furthermore, deletion of the Dbf4 protein-binding region in each Cdc7 mutant resulted in loss of growth inhibitory effect. Thus, dominant-negative effects of T281E, D182N, and D163N on growth can be best explained by inactivation of the wild-type Cdc7 function through titration of Dbf4 by these inactive kinases. Our results are consistent with the notion that association of Dbf4 with Cdc7 is essential for the G1 to S transition in S. cerevisiae.
Ohtoshi
A
A
Department of Molecular and Developmental Biology, Institute of Medical Science, The University of Tokyo, Minato-ku, Japan.
Miyake
T
T
Arai
K
K
Masai
H
H
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D004261:DNA Replication / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase; D015870:Gene Expression; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D013696:Temperature
D018797:Cell Cycle Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C489404:CDC7 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases