title: Cloning and biochemical characterization of Staphylococcus aureus type IA DNA topoisomerase comprised of distinct five domains.
abstract: DNA topoisomerases play critical roles in regulating DNA topology and are essential enzymes for cell survival. In this study, a gene encoding type IA DNA topoisomerase was cloned from Staphylococcus aureus (S. aureus) sp. strain C-66, and the biochemical properties of recombinant enzyme was characterized. The nucleotide sequence analysis showed that the cloned gene contained an open reading frame (2070 bp) that could encode a polypeptide of 689 amino acids. The cloned gene actually produced 79.1 kDa functional enzyme (named Sau-TopoI) in Escherichia coli (E. coli). Sau-TopoI enzyme purified from E. coli showed ATP-independent and Mg(2+)-dependent manners for relaxing negatively supercoiled DNA. The relaxation activity of Sau-TopoI was inhibited by camptothecin, but not by nalidixic acid and etoposide. Cleavage site mapping showed that the enzyme could preferentially bind to and cleave the sequence GGNN↓CAT (N and ↓ represent any nucleotide and cleavage site, respectively). All these results suggest that the purified enzyme is type IA DNA topoisomerase. In addition, domain mapping analysis showed that the enzyme was composed of conserved four domains (I through IV), together with a variable C-terminal region containing a unique domain V.
authors.affiliation: Department of Biotechnology, Chosun University, Gwangju 501-759, Republic of Korea.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Park
authors.forename: Hyun Ik
authors.initials: HI
authors.lastname: Kim
authors.forename: Jong Woo
authors.initials: JW
authors.lastname: Park
authors.forename: Jong Kun
authors.initials: JK
authors.lastname: Park
authors.forename: Jung Sup
authors.initials: JS
authors.lastname: Lee
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D059004:Topoisomerase I Inhibitors; D000255:Adenosine Triphosphate; D004264:DNA Topoisomerases, Type I