Purification and characterization of DNA topoisomerase V. An enzyme from the hyperthermophilic prokaryote Methanopyrus kandleri that resembles eukaryotic topoisomerase I.
DNA topoisomerase V is a novel prokaryotic enzyme related to eukaryotic topoisomerase I. The enzyme is a type I DNA topoisomerase and is recognized by polyclonal antibody against human topoisomerase I. We describe its purification from the hyperthermophilic methanogen Methanopyrus kandleri. The enzyme has high activity in crude extracts and is present in at least 1,500 copies/cell. Topoisomerase V migrates as a 110-kDa polypeptide in SDS-polyacrylamide gel electrophoresis and as a 142-kDa globular protein in gel filtration. It is active up to at least 100 degrees C on both positively and negatively supercoiled DNA and is not inhibited by single-stranded DNA. The enzyme works from 1 to 650 mM NaCl and up to 3.1 M potassium glutamate. It acts processively at low ionic strength and distributively at high NaCl or KCl concentration. Magnesium is not required and does not stimulate the enzymatic activity. Under DNA denaturing conditions, topoisomerase V catalyzes an unlinking reaction which results in substantial reduction in the linking number of closed circular DNA. The driving force for this process is DNA melting. Camptothecin is not nearly as good an inhibitor for topoisomerase V as it is for eukaryotic topoisomerase I. The unique occurrence of two major type I topoisomerases (reverse gyrase and topoisomerase V) in M. kandleri may shed new light on the evolution of this family of enzymes and supports the concept of a distant but significant relationship between some hyperthermophilic organisms and eukaryotes.
Slesarev
A I
AI
Molecular Biology Institute, University of California, Los Angeles 90024.
Lake
J A
JA
Stetter
K O
KO
Gellert
M
M
Kozyavkin
S A
SA
D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity
D004247:DNA; C082698:DNA topoisomerase V; D004264:DNA Topoisomerases, Type I