(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 22 April 2010 (22.04.2010) WO 2010/044101 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12N 9/90 (2006.0 1) C12Q 1/68 (2006.0 1) kind of national protection available): AE, AG, AL, AM, C12Q 1/18 (2006.01) GOlN 21/64 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12Q 1/533 (2006.01) GOlN 33/68 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/IN2009/000561 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 8 October 2009 (08.10.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 2159/MUM/2008 8 October 2008 (08. 10.2008) IN GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): V. B. TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, MEDICARE PVT. LTD. [IN/IN]; 141 Walchand Hirac- ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, hand Marg, Mumbai 400 001, Maharashtra (IN). MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, (72) Inventors; and ML, MR, NE, SN, TD, TG). (75) Inventors/Applicants (for US only): GUPTA, Munish- war, Nath [IN/IN]; Chemistry Department, Indian In sti Declarations under Rule 4.17: tute of Technology Delhi, Hauz Khas, New Delhi 110 016 — of inventorship (Rule 4Λ 7(iv)) (IN). SINGH, Pradeep, K. [IN/IN]; Chemistry Depart ment, Indian Institute of Technology Delhi, Hauz Khas, Published: New Delhi 110 016 (IN). RAGHAVA, Smita [IN/IN]; — with international search report (Art. 21(3)) Chemistry Department, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016 (IN). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agents: SAVANGIKAR, Vasant, Anantrao et al; Kr amendments (Rule 48.2(h)) ishna & Saurastri Associates, 74/F, Venus, Worli Sea Face, Mumbai 400 018, Maharashtra (IN). (54) Title: PURIFICATION AND ASSAY FOR TOPOISOMERASES AND USE OF THE ASSAY FOR SCREENING MODU LATORS OF TOPOISOMERASES (57) Abstract: This invention comprises a process of isolation and purification of Topoisomerases adsorbed on an immobilized metal affinity chromatography column charged with a metal ions by sequential elution by imidazole solution of different molar strengths. This invention also comprises a novel method of assay of Topoisomerase activity in presence or absence of a Topoiso- merase modulator and a kit for same wherein unreacted ScDNA separated from the product of topoisomerase action are removed using a chelating matrix charged with metal ions and fluorescence is reacted with a dye to result in a fluorescent DNA dye com plex, and measuring the florescence The assay may be performed in a multi-well plate for assay of Topoisomerase activity, for de tecting chemical agents for control of microorganisms or tumours or microbial spoilage of edible products by determining topoiso merase inhibiting or enhancing activity of a potential compound. The dye used may be a cyanine dye. TITLE PURIFICATION AND ASSAY FOR TOPOISOMERASES AND USE OF THE ASSAY FOR SCREENING MODULATORS OF TOPOISOMERASES. TECHNICAL FIELD This invention relates to isolation, -purification, and quantitative assay of DNA topoisomerase and screening of topoisomerase modulators including the protein CcdB using the assay BACKGROUND OF INVENTION DNA topoisomerases interconvert topoisomers of DNA. DNA topoisomerase I (Tl) and DNA topoisomerase Il [(TII) DNA gyrase] are enzymes which regulate the degree of supercoiling of intracellular DNA. There are other topoisomerases, e.g., Ill and IV which are related to I and Il and have similar function. These ubiquitous enzymes play a significant role in replication, transcription and recombination of DNA [1]. These enzymes are now well established as the targets for some of the antibiotics and anticancer drugs [2-5]. DNA topoisomerase I change the helical pitch of the genetic material by allowing passage of a DNA single strand through a transient nick created in the complementary strand of the double helix. Thus, a convenient assay for these enzymes for the purpose of screening antineoplastic drugs, kits for the assays and methods for isolation and purification of topoisomerases have become areas for work that serve a current need. PRIOR ART In some presently available methods for assay of enzyme activity of topoisomerases is measured in ε semiquantitative fashion by following the relaxation of supercoiled plasmid DNA (ScDNA) by agarose gel electrophoresis [6]. U.S. patent no. 5,998,152 describes both solid phase and liquid phase assay formats for measuring the activity of a topoisomerase in the presence of a potential topoisomerase activity modulator. This patent provides high-throughput screening methods, compositions, kits and integrated systems for performing the assays. In general, the assays are performed by contacting a nucleic acid with a topoisomerase in an appropriate reaction mixture that also includes a potential modulator of topoisomerase activity, a denaturant is added to the reaction mixture, resulting in the stabilization of any covalent complexes that are present, the presence or absence of topoisomerase-nucleic acid complex is then detected to ascertain whether a modulator of topoisomerase activity was present in the reaction mixture. WO2006/015369 deals with detection and continuous assay of topoisomerase activity spectroscopically wherein a fluorescent moiety is attached to the one strand of the duplex nucleic acid and fluorescent quencher is covalently attached to the complimentary strand of the duplex nucleic acid. The topoisomerase activity results in fluorescence from fluorescence moiety, which is measured spectroscopically. This principle is used to determine whether a compound is anti-viral, anti-cancer, antitrypanosome, anti-malarial, or antibacterial and the patent application also provides a kit for determining if a compound is a topoisomerase inhibitor comprising a duplex nucleic acid molecule with above mentioned properties, and instructions for use. In view of their biological importance, specifically in DNA replication and transcription, the enzymology of topoisomerases is an area of considerable interest. The purification of these enzymes has been a challenging task. For example, in case of Escherichia coli DNA gyrase [7] the subunits of the enzyme are separately isolated and the enzyme is obtained by reconstitution. Topoisomerases can either be isolated and purified from natural source or they can be isolated by cloning in a variety of host cells followed by ov3r-expression in the cloned cells and isolation there from. Topoisomerase I has often been purified by multistep purification protocol from various sources [8, 9], which often includes phosphocellulose/hydroxyapatite column chromatography followed by affinity chromatography on heparin affinity media [8, 9]. Topoisomerase I from a recombinant E. coli [10] has been purified by a shorter protocol using phosphocellulose chromatography. Topoisomerase Il is a dimer of heterodimer AB. Often B subunit has been purified on a novobiocin or monoclonal antibody affinity column and ieconstituted with gyrase A subunit which is purified again by a multistep protocol which includes ion exchange chromatography and phosphocellulose chromatography U]. In two cases, it has been possible to purify topoisomerase I and topoisomerase Il simultaneously [11,12]. Both protocols require fast protein liquid chromatography or high performance liquid chromatography. Above mentioned prior art methods of isolating topoisomerases are expensive and cumbersome approaches. SUMMARY OF INVENTION: In one embodiment this invention comprises a process of isolation and purification of a Topoisomerase from a cell free extract from lysed cells comprising the steps of (a) removing the nucleic acids, (b) adsorbing some proteins comprising Topoisomerases selectively on an immobilized metal affinity chromatography column charged with a metal ion, and (c) selectively and sequential elution of at Topoisomerases. The said Topoisomerases illustrated here are Topoisomerase I and Topoisomerase II. However, it is clear that it may be possible to separate otherTopoisomerases too by this method. In the illustrated embodiment of the process, the said metal ion is Zn2+ , however other metal ions such as Cu2+ may also give good results. In another aspect of the said process the said elution is done by using imidazole solution of different molar strengths selected such that each molar strength capable of dissolving one Topoisomerase. The process disclosed here of isolation and purification of a topoisomerase comprises steps of (a) lysing the bacterial cells with sonication, (b) centrifuging to get a crude lysate, (c) treating with streptomycin sulphate to remove