TOPO I (Human DNA Topoisomerase I, Wild Type)

• Species: Human
• Expression Host: Baculovirus-insect cell
• Tag: His-tag
• Purity: 90%
• Molecular Weight: 92.5 kDa.
• Gene Accession Number: NM_003286.

Purification and Quality ControL

The wild type DNA topoisomerase I protein (765 amino acids) was expressed in baculovirus system and purified by using an affinity column and FPLC chromatography. The purified TOPO I is greater than 90% homogeneous based on SDS-PAGE analysis.

Unit Definition (Activity)

1 unit equals 1 nanogram purified protein. 0.1 – 1 unit (ng) is sufficient for relaxing 0.5 µg of pBR322 supercoiled DNA in a 20 μl reaction.

Applications

Purified topo I has been used for in vitro transcriptional activation, pre-mRNA splicing/phosphorylation, DNA binding and DNA relaxation assays.

Formulation and Storage

The protein is in 20mM Tris-HCl pH7.9,100mM NaCl, 0.2mM EDTA, 1mM DTT and 20% glycerol. Stored at -70°C before use. Avoid repeated freeze thaw cycles.

Protein Sequence

SGDHLHNDSQ IEADFRLNDS HKHKDKHKDR EHRHKEHKKE KDREKSKHSN SEHKDSEKKH
KEKEKTKHKD GSSEKHKDKH KDRDKEKRKE EKVRASGDAK IKKEKENGFS SPPQIKDEPE
DDGYFVPPKE DIKPLKRPRD EDDADYKPKK IKTEDTKKEK KRKLEEEEDG KLKKPKNKDK
DKKVPEPDNK KKKPKKEEEQ KWKWWEEERY PEGIKWKFLE HKGPVFAPPY EPLPENVKFY
YDGKVMKLSP KAEEVATFFA KMLDHEYTTK EIFRKNFFKD WRKEMTNEEK NIITNLSKCD
FTQMSQYFKA QTEARKQMSK EEKLKIKEEN EKLLKEYGFC IMDNHKERIA NFKIEPPGLF
RGRGNHPKMG MLKRRIMPED IIINCSKDAK VPSPPPGHKW KEVRHDNKVT WLVSWTENIQ
GSIKYIMLNP SSRIKGEKDW QKYETARRLK KCVDKIRNQY REDWKSKEMK VRQRAVALYF
IDKLALRAGN EKEEGETADT VGCCSLRVEH INLHPELDGQ EYVVEFDFLG KDSIRYYNKV
PVEKRVFKNL QLFMENKQPE DDLFDRLNTG ILNKHLQDLM EGLTAKVFRT YNASITLQQQ
LKELTAPDEN IPAKILSYNR ANRAVAILCN HQRAPPKTFE KSMMNLQTKI DAKKEQLADA
RRDLKSAKAD AKVMKDAKTK KVVESKKKAV QRLEEQLMKL EVQATDREEN KQIALGTSKL
NYLDPRITVA WCKKWGVPIE KIYNKTQREK FAWAIDMADE DYEF

Background

Human DNA topoisomerase I (topo I) is a monomeric protein of 765 anino acids encoded by a single-copy gene (1-3). Based on its function, DNA topoisomerase I has been subdivided into four distinct domains. The N-terminal 214 amino acids of topo I comprise a highly charged N-terminal domain involved in protein-protein interactions with a number of cellular proteins. The highly conserved core domain expanded from amino acids 215 to 636 contains most catalytic residues and retains DNA binding activity. The active C-terminal domain (residues 713-765) is connected to the core domain by a poorly conserved linker domain (residues 636-712) (4-6).
Human DNA topoisomerase I is the best studied of the DNA topoisomerase family. It catalyzes the relaxation of both positive and negative supercoiled DNAs without the requirement of energy. In addition to DNA replication and transcriptional activation, DNA topoisomerase I also plays a major role in pre-mRNA splicing, cell cycle and other gene regulatory pathways during cell growth and development (7-10). Since topo I can cause large amounts of single-strand DNA breaks and this kind of DNA damage closely relates to most cancer, it is therefore believed that DNA topo I can be an important target for antitumor agents (11,12).

References

1. Liu, L. F. et al., (1981) Proc. Natl. Acad. Sci. USA 78 , 3487-3491
2. Juan, C.C. et al., (1988) Proc. Natl. Acad. Sci. USA 85, 8910-8913
3. D’Arpa, P. et al., (1988) Proc. Natl. Acad. Sci. USA 85, 2543-2547
4. Stewart, L. et al., (1996) J. Biol. Chem. 271, 7593-7601
5. Stewart, L. et al., (1996) J. Biol. Chem. 271, 7602-7608
6. Redinbo, M.R. et al., (1998) Science 279, 1504-1513
7. Pourquier, P. et al., (1997) J. Biol. Chem. 272, 26441-26447
8. Sekiguchi, J. et al., (1996) Proc. Natl. Acad. Sci. USA 93, 785-789
9. Kretzschmar, M. et al., (1993) Proc. Natl. Acad. Sci. USA 90, 11508-11512
10. Rossi, F. et al., (1996) Nature 381, 80-82
11. Giovanella, B.C. et al., (1989) Science 246, 1046-1048
12. Pommier, Y. et al., (1998) Biochem Biophys Acta 1400, 83-105

DISCLAIMER

This products is recommended For RESEARCH USE ONLY and is Not qualified for Use in Diagnostic or Therapeutic Procedures.