Analysis of Rad3 and Chk1 protein kinases defines different checkpoint responses.
Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses.


BACKGROUND
ATM/ATR/cell-cycle checkpoints/Chk1/Rad3
Martinho
R G
RG
MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, East Sussex, BN1 9RR, UK.
Lindsay
H D
HD
Flaggs
G
G
DeMaggio
A J
AJ
Hoekstra
M F
MF
Carr
A M
AM
Bentley
N J
NJ
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D016195:G2 Phase / Q000502:physiology; D018628:Gene Dosage; D006918:Hydroxyurea / Q000494:pharmacology; D007399:Interphase / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011836:Radiation Tolerance; D016196:S Phase / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D012641:Selection, Genetic; D013489:Suppression, Genetic; D014466:Ultraviolet Rays
D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D000071877:Checkpoint Kinase 1; C000605873:Chk1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; C061897:Rad3 protein, S cerevisiae; D004265:DNA Helicases; D006918:Hydroxyurea