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

The Journal of Biological Chemistry
C X ZhuYuk-Ching Tse-Dinh

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 degreesC, possibly accounting for the conservation of these residues in evolution.

References

May 1, 1991·Journal of Inorganic Biochemistry·P L Domanico, Y C Tse-Dinh
Jan 1, 1991·Critical Reviews in Biochemistry and Molecular Biology·R J Reece, A Maxwell
Oct 1, 1986·Journal of Bacteriology·G J PrussK Drlica
Oct 5, 1986·Journal of Molecular Biology·Y C Tse-Dinh, J C Wang
Jul 28, 1995·Science·R D FleischmannJ M Merrick
Oct 20, 1995·Science·C M FraserJ C Venter
Nov 1, 1995·Journal of Bacteriology·C M Joyce, T A Steitz
Jan 1, 1995·Annual Review of Biophysics and Biomolecular Structure·D B Wigley
Jan 1, 1995·Molecular Microbiology·P Polard, M Chandler
May 17, 1995·Biochimica Et Biophysica Acta·M GuptaY Pommier
Feb 1, 1995·Molecular Microbiology·A Luttinger
Jul 5, 1994·Proceedings of the National Academy of Sciences of the United States of America·T A Baker, L Luo
May 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·F ConfalonieriM Duguet
Jan 9, 1996·Proceedings of the National Academy of Sciences of the United States of America·R KrahM Gellert
Jan 1, 1995·Methods in Enzymology·V DerbyshireC M Joyce
Apr 16, 1996·Proceedings of the National Academy of Sciences of the United States of America·R HanaiJ C Wang
Jan 1, 1996·Annual Review of Biochemistry·J C Wang
Nov 15, 1996·Nucleic Acids Research·R HimmelreichR Herrmann
Jun 27, 1997·The Journal of Biological Chemistry·C X ZhuY C Tse-Dinh
Aug 1, 1997·Trends in Microbiology·Y C Tse-DinhR Menzel

❮ Previous
Next ❯

Citations

Jun 8, 2002·Journal of Molecular Biology·Christian G Noble, Anthony Maxwell
Oct 9, 2001·International Journal of Biological Macromolecules·C J Roche, Y C Tse-Dinh
Sep 28, 1998·Biochimica Et Biophysica Acta·Y C Tse-Dinh
Feb 13, 2003·Current Opinion in Structural Biology·Anita ChangelaAlfonso Mondragón
Nov 27, 1999·Structure·A Mondragón, R DiGate
Aug 30, 2008·Quarterly Reviews of Biophysics·Allyn J Schoeffler, James M Berger
Apr 13, 2011·Proceedings of the National Academy of Sciences of the United States of America·Zhongtao ZhangYuk-Ching Tse-Dinh
Aug 4, 2005·Nucleic Acids Research·Frédéric AllemandCiarán Condon
Dec 25, 2008·Nucleic Acids Research·Nicole M BakerAlfonso Mondragón
Jan 26, 2002·Molecular and Cellular Biology·Robert L DiazScott Keeney
Feb 3, 1999·Proceedings of the National Academy of Sciences of the United States of America·Q Liu, J C Wang
Mar 19, 2013·Annual Review of Biochemistry·Stefanie Hartman ChenTao-shih Hsieh
Feb 24, 2006·Journal of Molecular Biology·Paras Jain, Valakunja Nagaraja
Mar 6, 2002·The Journal of Biological Chemistry·John M LyleKarla Kirkegaard
Nov 9, 2001·The Journal of Biological Chemistry·Yong WangJames C Wang
Jan 26, 2002·The Journal of Biological Chemistry·Kay Perry, Alfonso Mondragón
Aug 20, 2014·The Journal of Biological Chemistry·Rakhi RajanAlfonso Mondragón
Jun 25, 2004·The Journal of Biological Chemistry·Bokun ChengYuk-Ching Tse-Dinh
Jan 9, 2004·The Journal of Biological Chemistry·Bokun ChengYuk-Ching Tse-Dinh
Oct 22, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Tumpa DasguptaYuk-Ching Tse-Dinh

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.