Polymerase eta and p53 jointly regulate cell survival, apoptosis and Mre11 recombination during S phase checkpoint arrest after UV irradiation.
Xeroderma pigmentosum variant (XPV) cells lack the damage-specific polymerase eta and undergo a protracted arrest at the S phase checkpoint(s) following UV damage. The S phase checkpoints encompass several qualitatively different processes, and stimulate downstream events that are dependent on the functional state of p53. Primary fibroblasts with wild-type p53 arrest in S, and require a functional polymerase eta (pol eta) to carry out bypass replication, but do not recruit recombination factors for recovery. XPV cells with non-functional p53, as a result of transformation by SV40 or HPV16 (E6/E7), recruit the hMre11/hRad50/Nbs1 complex to arrested replication forks, coincident with PCNA, whereas normal transformed cells preferentially use the pol eta bypass replication pathway. The formation of hMre11 foci implies that arrested replication forks rapidly undergo a collapse involving double strand breakage and rejoining. Apoptosis occurs after UV only in cells transformed by SV40, and not in normal or XPV fibroblasts or HPV16 (E6/E7) transformed cells. Conversely, ultimate cell survival in XPV cells was much less in HPV16 (E6/E7) transformed cells than in SV40 transformed cells, indicating that apoptosis was not a reliable predictor of cell survival. Inhibition of p53 transactivation by pifithrin-alpha or inhibition of protein synthesis by cycloheximide did not induce hMre11 foci or apoptosis in UV damaged fibroblasts. Inhibition of kinase activity with wortmannin did not increase killing by UV, unlike the large increase seen with caffeine. Since HPV16 (E6/E7) transformed XPV cells were highly UV sensitive and not further sensitized by caffeine, it appears likely that caffeine sensitization proceeds through a p53 pathway. The S phase checkpoints are therefore, a complex set of different checkpoints that are coordinated by p53 with the capacity to differentially modulate cell survival, apoptosis, bypass replication and hMre11 recombination.
Cleaver
J E
JE
UCSF Comprehensive Cancer Center, University of California, 2340 Sutter Street, Box 0808, Room N-424, San Francisco, CA 94143, USA. jcleaver@cc.ucsf.edu
Bartholomew
J
J
Char
D
D
Crowley
E
E
Feeney
L
L
Limoli
C L
CL
D017766:Acid Anhydride Hydrolases; D017209:Apoptosis / Q000502:physiology* / Q000528:radiation effects; D052160:Benzothiazoles; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology* / Q000528:radiation effects; D045643:DNA Repair Enzymes; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects; D006801:Humans; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D016196:S Phase / Q000502:physiology*; D013539:Simian virus 40 / Q000235:genetics; D013844:Thiazoles / Q000494:pharmacology; D014050:Toluene / Q000031:analogs & derivatives* / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000378:metabolism; D000099270:Y-Family DNA Polymerases
D017766:Acid Anhydride Hydrolases; D052160:Benzothiazoles; D018797:Cell Cycle Proteins; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins; D009856:Oncogene Proteins, Viral; D013844:Thiazoles; D014050:Toluene; D016159:Tumor Suppressor Protein p53; D001616:beta-Galactosidase; C121766:MRE11 protein, human; C112156:NBN protein, human; C121565:pifithrin; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C112158:RAD50 protein, human