Biochemical and immnulogical analysis of UV-induced photolesions

The induction and removal of UV-induced photolesions was investigated in confluent human fibroblasts employing two different approaches. Photolyase and highly purified E. coli Uvr A,B and C proteins were used to measure the frequency of 6-4 photoproducts (6-4PP) in transcriptionally active and inactive genes. At a UV-dose range of 20-60 J/m2 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m2 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m2, the repair of 6-4PP was very rapid in both active and inactive genes when compared to CPD removal. No strand specific repair of 6-4PP in active genes was observed, although repair of 6-4PP occurred preferentially in the active genes when compared to inactive X-chromosomal genes. In xeroderma pigmentosum group C cells 6-4PP were selectively removed from the transcribed strand of active genes. In these cells the kinetics of repair of CPD and 6-4PP from the transcribed strand of active genes was very similar. Our results indicate that 6-4PP can be removed by a transcription coupled repair pathway, but that repair of 6-4PP by the global repair system is much more efficient. The sam conclusion can be drawn from studies aimed to determine BUdR labelled repair sites in genomic fragments. The results of these studies indicate a lack of strandspecific repair of 6-4 PP in active genes in normal cells at a relative low UV-dose of 10J/m2