Purification and characterization of Thermus thermophilus UvrD.
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.
Collins
Ruairi
R
Department of Biochemistry, University College Cork, Cork, Ireland.
McCarthy
Tommie V
TV
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
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