Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase.
Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction.
Department of Tumor Biology, Institute of Biomedical Sciences, Iwate Medical University, Iwate 028-3694, Japan. syasuhir@iwate-med.ac.jp
Shinji
S
Yasuhira
Takeshi
T
Saito
Chihaya
C
Maesawa
Tomoyuki
T
Masuda
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004249:DNA Damage / Q000502:physiology*; D004265:DNA Helicases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000502:physiology*; D005720:Gamma Rays / Q000009:adverse effects; D017353:Gene Deletion; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D059767:Recombinational DNA Repair / Q000502:physiology*; D012568:Schizosaccharomyces / Q000502:physiology / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C480031:Rad11 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; C085412:rad52 protein, S pombe; 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; C456840:rad3 protein, S pombe; D004265:DNA Helicases; C099034:Rhp54 protein, S pombe