Transcription preferentially inhibits nucleotide excision repair of the template DNA strand in vitro

Abstract

It has been reported that pyrimidine dimers (pyrimidine mean value of pyrimidine) are removed preferentially from actively transcribing genes. Furthermore, the preferential repair is restricted to the transcribed strand of these genes. Currently there is no mechanistic explanation for these phenomena. In this study we investigated the effect of transcription on nucleotide excision repair using defined Escherichia coli systems consisting of DNA substrates containing a strong promoter and either (a) a T mean value of T at a defined position in the nontranscribed or transcribed strand or (b) photoproducts randomly distributed in both strands, as well as transcription and nucleotide excision repair enzymes. While a T mean value of T in the nontranscribed strand had no effect on transcription, a photodimer in the transcribed strand blocked transcription causing RNA polymerase to stall at the T mean value of T site. This stalled elongation complex inhibited the excision of the photodimer by (A)BC excinuclease resulting in a net effect of preferential repair of the nontranscribed strand in a mixture containing both substrates. Similarly, when we conducted transcription/repair experiments with a superhelical plasmid no enhanced repair of the transcribed gene was observed compared to nontranscribed regions. We conclude that RNA polymerase stalled at a photodimer does not direct the (A)BC excinuclease to the damaged template strand and therefore cannot account for the strand-specific repair observed in vivo.