Transcription-repair coupling determines the strandedness of ultraviolet mutagenesis in Escherichia coli.

Abstract

We have analyzed the spectra of UV-induced mutations in the Lad gene of a wild-type and an mfd strain of Escherichia coli. mfd strains have been recently proposed to be deficient in a factor coupling DNA repair and transcription. Analysis of UV-induced mutations occurring at adjacent pyrimidines showed that mutations in the wild-type strain arose largely from the nontranscribed strand but arose predominantly from the transcribed strand in the mfd strain. The overall strand switch was 14-fold. One mutation, G-C --AFT in the lacI initiation codon, showed a >300-fold shift. No effect was observed for mutations at non-pyrimidine-pyrimidine sequences. These results provide in vivo evidence for a key role of the mid gene in controlling the strandedness of mutagenesis and support the proposed role of the mfd gene product in directing DNA excision repair to the transcribed strand of a damaged gene.Heterogeneity in DNA repair, in prokaryotic and eukaryotic cells, has been observed at several levels: chromatin-domainspecific repair (1-3), preferential repair of active genes (4-10), and strand-specific repair (4,11,12). These different levels of repair specificity may be interrelated. Actively repaired chromatin domains often include actively transcribed genes, whereas most gene-specific repair may be explained by preferential repair of the transcribed strand of the gene. A current model for preferential repair of the transcribed strand stems from the observation that UV light-induced pyrimidine dimers, as well as other lesions, effectively block transcription (13,14). A stalled RNA polymerase-DNA complex may act as a signal to direct excision-repair enzymes to the transcribed strand of a gene, enhancing its repair (4). Precisely how this can be achieved is unclear, but one possibility is the existence of a transcription-repair coupling factor (TRCF). Recently, such a factor was purified from Escherichia coli using an in vitro strand-specific repair assay (15,16). This factor was able to direct nucleotide excisionrepair (UvrABC) to the transcribed strand ofa gene, resulting in preferential repair of this strand. Furthermore, a defect in transcription-stimulated repair was discovered in extracts from an E. coli mfd strain that could be complemented by the TRCF purified from wild-type cells (16).The mfd strain of E. coli was first characterized by Witkin (17) as a derivative unable to display the phenomenon of mutation frequency decline (MFD). MFD is the rapid loss of certain UV-induced nonsense suppressor mutations upon transient inhibition of protein synthesis following irradiation (17)(18)(19). This phenomenon was determined to be a manifestation of excision repair because (i) it was not observed in excision-repair-deficient (uvr) strains (17, 20) and (ii) the mfd strain displayed a reduced rate of excision repair (21,22). Bockrath and coworkers (23,24) noted that MFD only occurred when the lesion putatively responsible for the suppressor mutation resided on the transcribed strand of the tRNA gene, suggesting tha...

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

“…4). These results are consistent with studies that have demonstrated a moderate increase in UV sensitivity of cells defective in transcription-coupled repair such as mfd mutants of E. coli (23) (Fig. 4) and CS cells (24,25).…”

Rad23 Is Required for Transcription-Coupled Repair and Efficient Overall Repair in Saccharomyces cerevisiae

“…In a disruption mutant of RAD26, a marked reduction in TRC was observed in the RPB2 gene compared with that in wild-type (wt) cells while the removal of CPDs from the genome overall was unaffected (41). In addition, no marked increase in UV sensitivity was observed for the rad26 cells compared with wt cells (41), similar to what is observed with mfd mutant cells (9,28). Thus, it appears that RAD26 encodes a TRC factor in S. cerevisiae (41).…”

Nucleotide excision repair and photolyase preferentially repair the nontranscribed strand of RNA polymerase III-transcribed genes in Saccharomyces cerevisiae

“…Outside of this promoter region, however, in which slower repair was found, several mutagenesis hot spots were detected as in the pyrimidine clusters CTTT and CCCTCT at positions 54 and 89, respectively (Armstrong and Kunz 1990). Another example of higher mutagenesis in the TS was observed previously in mfd-deficient E. coli lacking the TCR process (Oller et al 1992). These results are in contrast with those of the TSs of RNAP II genes that are preferentially repaired and, consequently, show a lower mutagenesis as compared with the NTSs (Vrieling et al 1989;McGregor et al 1991;Sage et al 1993).…”