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Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase

Ogun Adebali,Yi‐Ying Chiou,Jinchuan Hu,A. Sancar,C. Selby

Published 2017 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance In transcription-coupled repair (TCR), nucleotide excision repair occurs most rapidly in the template strand of actively transcribed genes. TCR has been observed in a limited set of genes directly assayed in Escherichia coli cells. In vitro, Mfd translocase performs reactions necessary to mediate TCR: It removes RNA polymerase blocked by a template strand lesion and rapidly delivers repair enzymes to the lesion. This study applied excision repair sequencing methodology to map the location of repair sites in different E. coli strains. Results showed that Mfd-dependent TCR is widespread in the E. coli genome. Results with UvrD helicase demonstrated its role in basal repair, but no overall role in TCR. We used high-throughput sequencing of short, cyclobutane pyrimidine dimer-containing ssDNA oligos generated during repair of UV-induced damage to study that process at both mechanistic and systemic levels in Escherichia coli. Numerous important insights on DNA repair were obtained, bringing clarity to the respective roles of UvrD helicase and Mfd translocase in repair of UV-induced damage. Mechanistically, experiments showed that the predominant role of UvrD in vivo is to unwind the excised 13-mer from dsDNA and that mutation of uvrD results in remarkable protection of that oligo from exonuclease activity as it remains hybridized to the dsDNA. Genome-wide analysis of the transcribed strand/nontranscribed strand (TS/NTS) repair ratio demonstrated that deletion of mfd globally shifts the distribution of TS/NTS ratios downward by a factor of about 2 on average for the most highly transcribed genes. Even for the least transcribed genes, Mfd played a role in preferential repair of the transcribed strand. On the other hand, mutation of uvrD, if anything, slightly pushed the distribution of TS/NTS ratios to higher ratios. These results indicate that Mfd is the transcription repair-coupling factor whereas UvrD plays a role in excision repair by aiding the catalytic turnover of excision repair proteins.

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