The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining

Gagandeep Narula, Thirunavukkarasu Annamalai, Sandra Aedo, Bokun Cheng, Elena Sorokin, Agnes Wong, Yuk Ching Tse-Dinh

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3′-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.

Original languageEnglish (US)
Pages (from-to)18673-18680
Number of pages8
JournalJournal of Biological Chemistry
Volume286
Issue number21
DOIs
StatePublished - May 27 2011
Externally publishedYes

Fingerprint

Type I DNA Topoisomerase
Escherichia coli
DNA Cleavage
Arginine
Catalytic Domain
DNA
Phosphates
Yersinia pestis
Superhelical DNA
Bacterial DNA
Phosphotyrosine
Genetic Testing
Enzymes
Catalysis
Tyrosine
Metals
Ions
Phenotype
Mutation
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining. / Narula, Gagandeep; Annamalai, Thirunavukkarasu; Aedo, Sandra; Cheng, Bokun; Sorokin, Elena; Wong, Agnes; Tse-Dinh, Yuk Ching.

In: Journal of Biological Chemistry, Vol. 286, No. 21, 27.05.2011, p. 18673-18680.

Research output: Contribution to journalArticle

Narula, Gagandeep ; Annamalai, Thirunavukkarasu ; Aedo, Sandra ; Cheng, Bokun ; Sorokin, Elena ; Wong, Agnes ; Tse-Dinh, Yuk Ching. / The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining. In: Journal of Biological Chemistry. 2011 ; Vol. 286, No. 21. pp. 18673-18680.
@article{a76a15f14ab64c638ec5fee603bd3625,
title = "The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining",
abstract = "The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3′-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.",
author = "Gagandeep Narula and Thirunavukkarasu Annamalai and Sandra Aedo and Bokun Cheng and Elena Sorokin and Agnes Wong and Tse-Dinh, {Yuk Ching}",
year = "2011",
month = "5",
day = "27",
doi = "10.1074/jbc.M111.229450",
language = "English (US)",
volume = "286",
pages = "18673--18680",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "21",

}

TY - JOUR

T1 - The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining

AU - Narula, Gagandeep

AU - Annamalai, Thirunavukkarasu

AU - Aedo, Sandra

AU - Cheng, Bokun

AU - Sorokin, Elena

AU - Wong, Agnes

AU - Tse-Dinh, Yuk Ching

PY - 2011/5/27

Y1 - 2011/5/27

N2 - The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3′-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.

AB - The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3′-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.

UR - http://www.scopus.com/inward/record.url?scp=79956333418&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79956333418&partnerID=8YFLogxK

U2 - 10.1074/jbc.M111.229450

DO - 10.1074/jbc.M111.229450

M3 - Article

C2 - 21478161

AN - SCOPUS:79956333418

VL - 286

SP - 18673

EP - 18680

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 21

ER -