RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae.
The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53.
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
P R
PR
Dohrmann
G
G
Oshiro
M
M
Tecklenburg
R A
RA
Sclafani
D029681:Arabidopsis Proteins; D015152:Blotting, Northern; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics; D064447:Checkpoint Kinase 2; D003433:Crosses, Genetic; D004268:DNA-Binding Proteins / Q000235:genetics; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005820:Genetic Testing; D005838:Genotype; D006918:Hydroxyurea / Q000494:pharmacology; D015151:Immunoblotting; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics* / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; C104920:LSD1 protein, Arabidopsis; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011494:Protein Kinases; C489404:CDC7 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D001616:beta-Galactosidase; D006918:Hydroxyurea