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Literature summary for 5.6.2.1 extracted from

  • Narula, G.; Annamalai, T.; Aedo, S.; Cheng, B.; Sorokin, E.; Wong, A.; Tse-Dinh, Y.C.
    The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining (2011), J. Biol. Chem., 286, 18673-18680.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 5.6.2.1

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21AI Escherichia coli
expression of His-tagged Yersinia pestis topoisomerase I mutants, under the control of the BAD promoter in plasmid pYTOP, in Escherichia coli strain JD5 and purification by nickel affinity chromatography leading to a lethal phenotype of the Escherichia coli cells, overview Yersinia pestis

Protein Variants

Protein Variants Comment Organism
additional information expression of His-tagged Yersinia pestis topoisomerase I mutants, that are deficiency in DNA rejoining, under the control of the BAD promoter in plasmid pYTOP in Escherichia coli strain JD5 and purification by nickel affinity chromatography leading to a lethal phenotype of the Escherichia coli cells, overview Escherichia coli
R321F site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+ Escherichia coli
R321K site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+ Escherichia coli
R321L site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme, the mutant shows reduced DNA cleavage and requirement for Mg2+ Escherichia coli
R327F site-directed mutagenesis, the mutation leads to the SOS response and has a strong cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose, the mutant shows reduced DNA cleavage and requirement for Mg2+ Yersinia pestis
R327I site-directed mutagenesis, the mutation leads to the SOS response and has a strong cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose, the mutant shows reduced DNA cleavage and requirement for Mg2+ Yersinia pestis
R327L site-directed mutagenesis, the mutation leads to the SOS response and has a strong cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose, the mutant shows reduced DNA cleavage and requirement for Mg2+ Yersinia pestis
R327S naturally occuring mutation identified in the AW3-75 mutant, the mutation leads to the SOS response and has a cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose Yersinia pestis
R327S site-directed mutagenesis, the mutant does not show a phenotype Yersinia pestis
R327W site-directed mutagenesis, the mutation leads to the SOS response and has a strong cell killing effect when the mutant is overexpressed in Escherichia coli and induced with 0.2% arabinose, the mutant shows reduced DNA cleavage and requirement for Mg2+ Yersinia pestis
T265M site-directed mutagenesis, the mutant does not show a phenotype Yersinia pestis
V559I site-directed mutagenesis, the mutant does not show a phenotype Yersinia pestis
Y319F site-directed mutagenesis, constants of oligonucleotide binding compared to the wild-type enzyme Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
nucleus
-
 Escherichia coli 5634
-
nucleus
-
 Yersinia pestis 5634
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ wild-type EcTOP1 does not require Mg2+ for DNA cleavage, but R321 mutants do Escherichia coli
Mg2+ wild-type YpTOP1 does not require Mg2+ for DNA cleavage, but R327 mutants do Yersinia pestis

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-
Yersinia pestis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged topoisomerase I mutants from Escherichia coli strain JD5 and purification by nickel affinity chromatography Yersinia pestis
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21AI by nickel affinity chromatography Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA. DNA cleavage can still take place in the absence of Arg231 function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate, but DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. 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 Escherichia coli ?
-
 ?
additional information the strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA. DNA cleavage can still take place in the absence of Arg237 function if Mg2+ is present to enhance the interaction of the enzyme with the scissile phosphate, but DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. 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 Yersinia pestis ?
-
 ?

Synonyms

Synonyms Comment Organism
Topoisomerase I
-
 Escherichia coli
Topoisomerase I
-
 Yersinia pestis
type IA topoisomerase
-
 Escherichia coli
type IA topoisomerase
-
 Yersinia pestis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Escherichia coli
37
-
 assay at Yersinia pestis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at Escherichia coli
8
-
 assay at Yersinia pestis