title: Purification and characterization of Thermus thermophilus UvrD.
abstract: The DNA helicase UvrD (helicase II) protein plays an important role in nucleotide excision repair, mismatch repair, rolling circular plasmid replication, and in DNA replication. A homologue of the Escherichia coli uvrD gene was previously identified in Thermus thermophilus; however, to date, a UvrD helicase has not been purified and characterized from a thermophile. Here we report the purification and characterization of a UvrD protein from Thermus thermophilus HB8. The purified UvrD has a temperature range from 10 degrees to >65 degrees C, with an optimum of 50 degrees C, within the temperature limits of the assay. The enzyme had a requirement for divalent metal ions and nucleoside triphosphates which related to enzyme activity in the order ATP > dATP > dGTP > GTP >> CTP > dCTP >> UTP. A simple real-time helicase assay was developed that should facilitate detailed kinetic studies of the enzyme. Evaluation of helicase substrates using this assay showed that the enzyme was highly active on a double-stranded DNA with 5' recessed ends in comparison with substrates with 3' recessed or blunt ends, and supports enzyme translocation in a 3'-5' direction relative to the strand bound by the enzyme.
authors.affiliation: Department of Biochemistry, University College Cork, Cork, Ireland.
authors.forename: Ruairi
authors.initials: R
authors.lastname: Collins
authors.forename: Tommie V
authors.initials: TV
authors.lastname: McCarthy
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002021:Buffers; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004277:DNA, Single-Stranded / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D002021:Buffers; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases