A ROLE FOR P53 IN DNA END REJOINING BY HUMAN CELL-EXTRACTS

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same dono r, TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and es sentially no p53 protein, respectively. Total cellular extracts from T K6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that t he major products formed were linear multimers. There was approximatel y 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts comp ared with TK6 extracts. Total DNA from end-rejoining reactions was pur ified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no sig nificant difference between the three cell lines, Gel analysis reveale d that misrepair involved only deletions. Sequence analysis of ii misr epaired products from each cell line showed 12 different deletions fro m 4 to 48 bp in length, but each cell line yielded similar product typ es. These results indicate that total cellular extracts from human lym phoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressin g wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events. (C) 1997 Elsevier Science B.V.