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J. Biol. Chem., Vol. 277, Issue 7, 4959-4965, February 15, 2002

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The Domain Organization and Properties of Individual Domains of DNA Topoisomerase V, a Type 1B Topoisomerase with DNA Repair Activities^*

Galina I. Belova^§, Rajendra Prasad^§, Igor V. Nazimov, Samuel H. Wilson, and Alexei I. Slesarev^¶

From the  M. M. Shemyakin and Yu A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117871 Moscow, Russia, the ^§ Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, and the ^¶ Fidelity Systems, Inc., Gaithersburg, Maryland 20879

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 °C and 80 °C. The Topo V protein is comprised of (i) a 44-kDa NH₂-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 NH₂-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.

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