title: The domain organization and properties of individual domains of DNA topoisomerase V, a type 1B topoisomerase with DNA repair activities.
abstract: Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.
authors.affiliation: M. M. Shemyakin and Yu A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow, Russia.
authors.forename: Galina I
authors.initials: GI
authors.lastname: Belova
authors.forename: Rajendra
authors.initials: R
authors.lastname: Prasad
authors.forename: Igor V
authors.initials: IV
authors.lastname: Nazimov
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
authors.forename: Alexei I
authors.initials: AI
authors.lastname: Slesarev
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000494:pharmacology; D013696:Temperature; D013820:Thermolysin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D012492:Salts; D014443:Tyrosine; D004247:DNA; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D013820:Thermolysin; C082698:DNA topoisomerase V; D004264:DNA Topoisomerases, Type I