P53-MEDIATED TRANSCRIPTION INDUCES RESISTANCE OF DNA TO UV INACTIVATION

A possible role of p53-dependent transcription in the induction of DIV A repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p 53 negative-mouse cell line [(10)1], When a p53-expressing plasmid (pS V.p53) was cotransfected into these cells, CAT expression levels persi sted even after prolonged UV irradiation. In comparison, CAT expressio n from pSV2.CAT, which lacks a p53-responsive element in its SV40 prom oter, dropped off much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was obs erved with three other constructs when the reporter gene was under the control of the ras, beta-actin or fos promoter. Mouse cells (A1-5) th at constitutively express a temperature-sensitive mutant (135 AV) of m ouse p53 also generated, at 32 degrees C, higher levels of enzyme expr essed from UV-irradiated pRGC,FOS,CAT than from UV-irradiated pSV2.CAT . The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC ,FOS,CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribut ion for various times of UV-irradiation, The observed relative CAT exp ression levels from irradiated pSV2,CAT and pRGC,FOS,CAT in the absenc e of p53 were consistent with those numbers. These results show that W T p53-mediated transcription directs a resistance of the transcribed D NA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near nor mal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants w ith reduced transcriptional activity retained this ability. This indic ates that although resistance to UV inactivation is transcriptionally- dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.