TOPO I –mt (Human DNA Topoisomerase I, Y723F)

• Species: Human
• Expression Host: Baculovirus-insect cell
• Tag: His-tag
• Purity: 90%
• Molecular Weight: 92.4 kDa.
• Gene Accession Number: NM_003286.

Purification and Quality Control

The His-tag recombinant protein is purified by affinity chromatography in combination with FPLC columns. The purified TOPO I –mt (Y723F) is greater than 95% homogeneous based on SDS-PAGE analysis.

Unit Definition (Activity)

1 unit equals 1 nanogram of purified protein. 1 unit is sufficient for a gel mobility shift assay in a 20 µl reaction; 100 units are sufficient for protein-protein interaction assays.

Applications

Purified topo I (Y723F) has been tested for in vitro DNA relaxation assay. 0.1 - 1 unit (ng) of mutated topo I (Y723F) are enough for testing in relaxation activity.

Formulation and Storage

The protein is in 20mM Tris-HCl pH8.0,100mM 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 NFLDPRITVA WCKKWGVPIE KIYNKTQREK FAWAIDMADE DYEF

Background

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 (1,2). Tyrosine 723 was identified as an active site for the DNA binding activity of DNA topoisomerase I. The covalent intermediate of topo I and DNA complex includes nucleophilic attack by the O4-oxygen of tyrosine 723 on a phosphester linkage in the DNA (3-5). Mutation from tyrosine to phenylalanine at position 723 preferentially binds the supercoiled DNA rather than relaxed DNA in the mixture of supercoiled and relaxed DNAs (6). But mutation at tyr723 neither affects its kinase activity that phosphorylates splicing factors of SR protein family (7) nor its transcription activity of class II genes in vitro (8).
The mutant His tagged Y723F of DNA topoisomerase I protein (Y723F) was expressed in a baculovirus system and purified by affinity and FPLC chromatography.

References

1. Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375
2. Champoux, J.J. (2001) Annu. Rev. Biochem 70, 369-413
3. Champoux, J.J. (1981) J. Biol. Chem. 256, 4805-4809
4. Madden, K.R. et al., (1992) Cancer Res. 52, 525-532
5. Lynn, R.M. et al., (1989) Proc. Natl. Acad. Sci. USA 86, 3559-3563
6. Madden, K.R. et al., (1996) EMBO J. 14, 5399-5404
7. Rossi, F. et al., (1998) Nucleic Acids Res. 26, 2963-2970

8. Wang, Z. et al., (1998) Mol. Cell 1, 749-757

DISCLAIMER

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