Induction and repair of cyclobutane pyrimidine dimers in the Escherichia coli tRNA gene tyrT:Fis protein affects dimer induction in the control region and suppresses preferential repair in the coding region of the transcribed strand, except in a short region near the transcription start site

Abstract

We analysed induction and repair of UV induced pyrimidine dimers in the Escherichia coli tRNA gene tyrT. In wild-type (WT) log or stationary phase different patterns of induction occurred in the three Fis binding sites and the core promoter −35 sequence of the control region: this was absent in fis − cells. In stationary WT cells, slow, similar rates of repair occurred throughout the non-transcribed strand (NTS). Faster repair occurred in the NTS control region in WT log phase. NTS repair in fis − cells was similar, except the control region differed less between phases. Heterogeneous repair occurred along the transcribed strand (TS). In the control region repair was faster than in the NTS. Repair in the TS coding region changed between growth phases or if repair took place in different media. When irradiated log phase WT cells were in rich medium, two TS domains were evident: a fast-repaired domain within 31 nucleotides from the transcription start site; and a more slowly repaired domain composed of the rest of the TS. A sharp gradient existed in the small domain with very fast repair at the beginning and diminished repair towards the end. Fast transcription coupled repair (TCR) in the small domain was absent in the TS large domain, where repair was similar to the NTS and to the entire TS in mfd − cells. In similarly treated stationary phase WT cells, TCR occurred in the large domain. Depletion of Fis reinstates TCR to a lesser extent, whilst a substitution of five nucleotides at the Fis binding sites in the upstream activating sequence reinstates TCR. Reinstatement of TCR was also achieved by incubating irradiated WT cells in minimal salt medium without the required amino acid. Our results suggest that Fis indirectly suppresses preferential repair in the TS large domain by stimulating transcription.