Effect of wild-type, S15D and R175H p53 proteins on DNA end joining in vitro: potential mechanism of DNA double-strand break repair modulation.
Balanced regulation of DNA double-strand break (DSB) repair is crucial for genetic integrity and cell survival. Cells perform DSB repair either by homologous recombination (HR) or by non-homologous end joining (NHEJ). Either option carries risk for DNA instability. The presence in the cell of the tumour suppressor p53 has been shown to suppress the levels of HR; however, the effect of p53 on DNA EJ is less well understood. Here we demonstrate dramatically increased DNA EJ activity in cell-free extracts from p53(-/-) mouse embryo fibroblasts (MEFs) compared with p53(+/+) MEFs. The addition of wild-type (wt) p53 to p53(-/-) MEFs extracts inhibited DNA EJ in a dose-dependent manner. Binding of wt p53 to DNA ends in vitro protected them from exonuclease attack and inhibited T4 DNA ligase-dependent EJ. This inhibitory effect was markedly enhanced for p53 R175H, a cancer-derived mutant of p53. In contrast, inhibition was negated in the presence of p53 S15D, a phosphorylation-mimicking mutant protein. Interestingly, p53 S15D stimulated in vitro DNA EJ of the blunt-ended DNA by T4 DNA ligase. Here we discuss the possibility that, in conjunction with its ability to control levels of HR, p53 may also serve to suppress DNA EJ in cells under normal conditions. This suppression may be associated with DNA-dependent protein kinases or ATM kinases, providing potential crosstalk between major cellular pathways of DNA repair and cell-cycle checkpoint mechanisms.
Okorokov
Andrei L
AL
YCR p53 Research Group, Department of Biology, University of York, York, YO10 5DD, UK. a.okorokov@mail.cryst.bbk.ac.uk
Warnock
Lorna
L
Milner
Jo
J
D000483:Alleles; D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D005347:Fibroblasts / Q000378:metabolism; D016158:Genes, p53; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism* / Q000502:physiology
D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D011088:DNA Ligases