Transcription-modulated repair in Escherichia coli evident with UV-induced mutation spectra in supF

We have determined several mutation spectra with the supF sequence after UV mutagenesis in Escherichia coli. The cells were either mfd(+) or mfd(-) and grown in defined or complex medium. The tRNA supF gene was expressed from the plasmid pZ189 or pLS1D (similar to pLS189, a variant of pZ189, but with a tac promoter for supF). Most of the mutations with either plasmid could be attributed to possible targeting photoproducts at dipyrimidine sites in the transcribed (TS) or non-transcribed (NTS) DNA strand with differential characteristics relevant to the repair process "mutation frequency decline" (MFD): (1) with pZ189, targeting sites in TS were favored over sites in NTS in all conditions except after an explicit MFD incubation with mfd(+) cells, when there was a majority in NTS; (2) with pLS1D (tac promoter), there was always a marked bias for targeting sites in TS and this was not altered by an MFD incubation; and (3) with pLS1D, spectra with mfd(-) cells vis-à-vis wild-type indicated a notable shift in the position of a hot-spot (both targeting sites in TS) and an increase in deletion mutations. The results support the Selby-Sancar idea that transcription-coupled nucleotide excision repair (TCR) at tRNA genes accounts for MFD and can be inhibited by rapid transcription. During interference of TCR by rapid transcription, however, the presence or absence of functional Mfd protein (transcription-repair coupling factor) can still influence the pattern of mutation, e.g. alter the position of a hot-spot in pLS1D. Only when a tRNA promoter is modulated by an MFD condition is transcription at a rate conducive to TCR. There were several deletion mutations with pLS1D between direct repeats (not present in pZ189) and a model for their production by UV damage is suggested. The spectra with pZ189 in E. coli had similarities with those published for UV mutagenesis in human cells, e.g. mutations at positions approximately 124 and 156.