Transcription-coupled nucleotide excision repair of a gene transcribed by bacteriophage T7 RNA polymerase in Escherichia coli.
Transcription-coupled nucleotide excision repair (TC-NER) removes certain kinds of lesions from the transcribed strand of expressed genes. The signal for TC-NER is thought to be RNA polymerase stalled at a lesion in the DNA template. In Escherichia coli, the stalled polymerase is dissociated from the lesion by the transcription repair coupling factor (Mfd protein), which also recruits excision repair proteins to the site resulting in efficient removal of the lesion. TC-NER has been documented in cells from a variety of organisms ranging from bacteria to humans. In each case, the RNA polymerase involved has been a multimeric protein complex. To ascertain whether a gene transcribed by the monomeric RNA polymerase of bacteriophage T7 could be repaired by TC-NER, we constructed strains of E. coli in which the chromosomal lacZ gene is controlled by a T7 promoter. In the absence of T7 RNA polymerase, little or no beta-galactosidase is produced, indicating that the E. coli RNA polymerase does not transcribe lacZ efficiently, if at all, in these strains. By introducing a plasmid (pAR1219) carrying the T7 gene 1 under control of the E. coli lac UV5 promoter into these strains, we obtained derivatives in which the level of T7 RNA polymerase could be regulated. In cultures containing upregulated levels of the polymerase, beta-galactosidase was actively produced indicating that the T7 RNA polymerase transcribes the lacZ gene efficiently. Under these conditions, we observed that UV-induced cyclobutane pyrimidine dimers were removed more rapidly from the transcribed strand of lacZ than from the nontranscribed strand, supporting the conclusion that TC-NER occurred in this gene. This response was absent in an mfd-1 mutant, indicating that the underlying mechanism may be similar to that for the bacterial RNA polymerase.
Ganesan
Ann K
AK
Department of Biology, Stanford University, Stanford, CA 94305-5020, USA. anngan@stanford.edu
Hanawalt
Philip C
PC
D001483:Base Sequence; D017931:DNA Primers; D004260:DNA Repair; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases