Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I

Chang Xi Zhu, Camille J. Roche, Nikolaos Papanicolaou, Anna DiPietrantonio, Yuk Ching Tse-Dinh

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42 °C, possibly accounting for the conservation of these residues in evolution.

Original languageEnglish (US)
Pages (from-to)8783-8789
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number15
DOIs
StatePublished - Apr 10 1998
Externally publishedYes

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arginine glutamate
Type I DNA Topoisomerase
Mutagenesis
Site-Directed Mutagenesis
Aspartic Acid
Escherichia coli
Catalytic Domain
DNA
DNA Cleavage
Hydroxyl Radical
Mutation
Enzymes
DNA Topoisomerases
Acidic Amino Acids
Superhelical DNA
Basic Amino Acids
Type II DNA Topoisomerase
Phosphotyrosine
Catalysis
Alanine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I. / Zhu, Chang Xi; Roche, Camille J.; Papanicolaou, Nikolaos; DiPietrantonio, Anna; Tse-Dinh, Yuk Ching.

In: Journal of Biological Chemistry, Vol. 273, No. 15, 10.04.1998, p. 8783-8789.

Research output: Contribution to journalArticle

Zhu, Chang Xi ; Roche, Camille J. ; Papanicolaou, Nikolaos ; DiPietrantonio, Anna ; Tse-Dinh, Yuk Ching. / Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 15. pp. 8783-8789.
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