Direct involvement of the tumor suppressor p53 in nucleotide excision repair

The tumor suppressor p53 enhances repair of UVC-induced DNA damage. The comet-NE assay, a conventional alkaline comet assay which includes a nuclear digestion step, was used to examine the effects of p53 on the excision activity of nuclear extracts (NEs). In contrast with untreated NEs, NEs immunodepleted of p53 or NEs of p53-null cells were unable to excise UVC-induced DNA adducts. Introduction of p53 by transfection restored the excision activity to NEs of p53-null cells. Deletion of the N-terminal 99 amino acids and/or the C-terminal 85 amino acids of p53 barely affected the excision activity, whereas further deletion of the C-terminus of p53 by another 10 amino acids completely abolished the excision activity of NEs. Immunostaining following localized UV irradiation was used to examine the effects of p53 on the recruitment of repair proteins for nucleotide excision repair (NER). Although recruitment of XPC occurred regardless of the presence of p53, the recruitment of XPB was p53-dependent. However, p53 with the 95 amino acid deletion at its C-terminus was unable to support this recruitment of XPB. Consistently, intact p53 (but not the C-terminal 95 residue truncated version) was detected in co-immunoprecipitation assays with an anti-XPB antibody. These results support the hypothesis that p53 facilitates NER through direct involvement by protein–protein interactions.