Effects of modification of the active site tyrosine of human DNA topoisomerase I

Rong Gao, Yi Zhang, Larisa Dedkova, Ambar K. Choudhury, Nicolas J. Rahier, Sidney M. Hecht

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

The human topoisomerase I-mediated DNA relaxation reaction was studied following modification of the enzyme at the active site tyrosine (position 723). A series of unnatural tyrosine analogues was incorporated into the active site of human topoisomerase I by utilizing misacylated suppressor tRNAs in an in vitro protein synthesizing system. The relaxation activities of the modified human topoisomerase I analogues having varied steric, electronic, and stereochemical features were all greatly diminished relative to that of the wild type. It was found that modifications involving replacement of the nucleophilic tyrosine OH group with NH2, SH, or I groups eliminated DNA relaxation activity, as did changing the orientation of the nucleophilic tyrosine OH group. Only tyrosine analogues having the phenolic OH group in the normal position with respect to the protein backbone were active; the relative activities could be rationalized in chemical terms on the basis of the H-bonding and the electronic effects of the substituents attached to the meta position of the aromatic ring. In addition, the poisoning of one of the modified human topoisomerase I analogues, as part of covalent binary complexes with DNA, by CPT and 20-thio CPT was evaluated.

Original languageEnglish (US)
Pages (from-to)8402-8410
Number of pages9
JournalBiochemistry
Volume45
Issue number27
DOIs
StatePublished - Jul 11 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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