The limitations of the G1-S checkpoint.
It has been proposed that the G(1)-S checkpoint is the critical regulator of genomic stability, preventing the cell cycle progression of cells with a single DNA double-strand break. Using fluorescence-activated cell sorting analysis of asynchronous cells and microscopic analysis of asynchronous and synchronized cells, we show that full blockage of S-phase entry is only observed >4 hours after irradiation. The process is ataxia-telangiectasia mutated (ATM) dependent and Chk1/2 independent and can be activated throughout G(1) phase. By monitoring S-phase entry of irradiated synchronized cells, we show that the duration of arrest is dose dependent, with S-phase entry recommencing after arrest with kinetics similar to that observed in unirradiated cells. Thus, G(1)-S checkpoint arrest is not always permanent. Following exposure to higher doses (> or =2 Gy), G(1)-S arrest is inefficiently maintained, allowing progression of G(1)-phase cells into G(2) with elevated gammaH2AX foci and chromosome breaks. At early times after irradiation (< or =4 h), G(1)-S checkpoint arrest is not established but cells enter S phase at a reduced rate. This early slowing in S-phase entry is ATM and Chk2 dependent and detectable after 100 mGy, showing a novel and sensitive damage response. However, the time needed to establish G(1)-S checkpoint arrest provides a window when cells can progress to G(2) and form chromosome breaks. Our findings detail the efficacy of the G(1)-S checkpoint and define two significant limitations: At early times after IR, the activated checkpoint fails to efficiently prevent S-phase entry, and at later times, the checkpoint is inefficiently maintained.
Deckbar
Dorothee
D
Radiation Biology and DNA Repair, Darmstadt University of Technology, Darmstadt, Germany.
Stiff
Thomas
T
Koch
Barbara
B
Reis
Caroline
C
Löbrich
Markus
M
Jeggo
Penny A
PA
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000502:physiology; D002455:Cell Division / Q000235:genetics / Q000528:radiation effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D019457:Chromosome Breakage; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology / Q000528:radiation effects; D005434:Flow Cytometry; D016193:G1 Phase / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D016195:G2 Phase / Q000235:genetics / Q000528:radiation effects; D006657:Histones / Q000502:physiology; D006801:Humans; D007259:Infrared Rays; D011494:Protein Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D016196:S Phase / Q000235:genetics / Q000502:physiology* / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000502:physiology
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases