Genome-wide transcription-coupled repair inEscherichia coliis mediated by the Mfd translocase.
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 inEscherichia coliNumerous 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 ofuvrDresults 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 ofmfdglobally 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 ofuvrD, 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.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260.
Ogun
O
Adebali
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260.
Yi-Ying
YY
Chiou
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260.
Jinchuan
J
Hu
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260 aziz_sancar@med.unc.edu cselby@med.unc.edu.
Aziz
A
Sancar
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260 aziz_sancar@med.unc.edu cselby@med.unc.edu.
Christopher P
CP
Selby
D001426:Bacterial Proteins / Q000378:metabolism*; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D055106:Genome-Wide Association Study; D059014:High-Throughput Nucleotide Sequencing; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C072103:transcription repair coupling factor protein, Bacteria; D002945:Cisplatin
Mfd; UvrD; XR-seq; mutagenesis; transcription-coupled repair