The complex choreography of transcription-coupled repair.

A quarter of a century has elapsed since the discovery of transcription-coupled repair (TCR), and yet our fascination with this process has not diminished. Nucleotide excision repair (NER) is a versatile pathway that removes helix-distorting DNA lesions from the genomes of organisms across the evolutionary scale, from bacteria to humans. TCR, defined as a subpathway of NER, is dedicated to the repair of lesions that, by virtue of their location on the transcribed strands of active genes, encumber elongation by RNA polymerases. In this review, we will report on newly identified proteins, protein modifications, and protein complexes that participate in TCR in Escherichia coli and in human cells. We will discuss general models for the biochemical pathways and how and when cells might choose to utilize TCR or other pathways for repair or bypass of transcription-blocking DNA alterations.

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.

Transcription coupled nucleotide excision repair in Escherichia coli can be affected by changing the arginine at position 529 of the beta subunit of RNA polymerase.

The proposed mechanism for transcription coupled nucleotide excision repair (TCR) invokes RNA polymerase (RNAP) blocked at a DNA lesion as a signal to initiate repair. In Escherichia coli, TCR requires the interaction of RNAP with a transcription-repair coupling factor encoded by the mfd gene. The interaction between RNAP and Mfd depends upon amino acids 117, 118, and 119 of the beta subunit of RNAP; changing any one of these to alanine diminishes the interaction [1]. Using direct assays for TCR, and the lac operon of E. coli containing UV induced cyclobutane pyrimidine dimers (CPDs) as substrate, we have found that a change from arginine to cysteine at amino acid 529 of the beta subunit of the RNAP inactivates TCR, but does not prevent the interaction of RNAP with Mfd. Our results suggest that this interaction may be necessary but not sufficient to facilitate TCR.