Initiation of transcription-coupled repair characterized at single-molecule resolution

• Published in: Nature (London) Vol. 490; no. 7420; pp. 431 - 434
• Main Authors: Howan, Kévin, Smith, Abigail J., Westblade, Lars F., Joly, Nicolas, Grange, Wilfried, Zorman, Sylvain, Darst, Seth A., Savery, Nigel J., Strick, Terence R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.10.2012; Nature Publishing Group
• Subjects: 631/337/1427; 631/337/572; Adenosine triphosphate; Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; ATP; Bacterial Proteins - metabolism; Biocatalysis; Biodiversity; Biological and medical sciences; Biological Physics; Deoxyribonucleic acid; Development Biology; DNA; DNA Damage; DNA Repair; DNA-Directed RNA Polymerases - metabolism; E coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic transcription; Humanities and Social Sciences; Hydrolysis; Kinetics; Lesions; letter; Life Sciences; Molecular and cellular biology; Molecular genetics; multidisciplinary; Mutagenesis. Repair; Physics; Physiological aspects; Populations and Evolution; Promoter Regions, Genetic - genetics; Proteins; Pyrimidine Dimers - chemistry; Pyrimidine Dimers - metabolism; RNA polymerase; Science; Science (multidisciplinary); Transcription Elongation, Genetic; Transcription Factors - metabolism; Transcription Initiation, Genetic; Transcription Termination, Genetic; Transcription, Genetic; United States; United Kingdom; Repair; Transcription initiation
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature11430

The early stages of transcription-coupled DNA repair are observed at single-molecule resolution; the Escherichia coli DNA translocase molecule Mfd is shown to promote RNA polymerase dissociation by catalysing two irreversible, ATP-dependent transitions. Two-step process to restart DNA polymerase The presence of a DNA lesion stalls elongating RNA polymerase, and so that transcription can resume a process known as transcription-coupled repair is initiated. This involves a bacterial accessory factor, Mfd, that promotes release of the stalled polymerase prior to removal of the lesion by DNA-repair factors. This study follows the early stages of transcription-coupled repair using a single-molecule approach. Mfd is shown to promote polymerase dissociation in two stages: it first drives partial collapse of the transcription bubble to form a long-lived intermediate, then a series of irreversible steps wrenches the polymerase off the DNA and restores it to normal B-form. These experiments thus reveal generic kinetic features of transcription-coupled repair initiation that should provide a framework for understanding the process in vivo . Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase 1 , 2 , 3 . Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.