title: The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.
abstract: During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of the mechanism by which UvrD disassembles the post-incision complex during NER. In further experiments we showed that PcrA helicase from Bacillus stearothermophilus can also displace UvrC and the excised oligonucleotide from a post-incision NER complex, which supports the idea that PcrA performs a UvrD-like function during NER in gram-positive organisms.
authors.affiliation: DNA-protein Interactions Unit, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: Manelyte
authors.forename: Colin P
authors.initials: CP
authors.lastname: Guy
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Smith
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dillingham
authors.forename: Peter
authors.initials: P
authors.lastname: McGlynn
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Savery
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D004247:DNA; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases