title: Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface.
abstract: Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.
authors.affiliation: Howard Hughes Medical Institute, The Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Deaconescu
authors.forename: Anastasia
authors.initials: A
authors.lastname: Sevostyanova
authors.forename: Irina
authors.initials: I
authors.lastname: Artsimovitch
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Grigorieff
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D018360:Crystallography, X-Ray; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D012318:RNA Polymerase I / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C058093:UvrB protein, E coli; C072103:transcription repair coupling factor protein, Bacteria; D000255:Adenosine Triphosphate; D012318:RNA Polymerase I; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases