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 Mg(2+) 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: Gagandeep
authors.initials: G
authors.lastname: Narula
authors.forename: Thirunavukkarasu
authors.initials: T
authors.lastname: Annamalai
authors.forename: Sandra
authors.initials: S
authors.lastname: Aedo
authors.forename: Bokun
authors.initials: B
authors.lastname: Cheng
authors.forename: Elena
authors.initials: E
authors.lastname: Sorokin
authors.forename: Agnes
authors.initials: A
authors.lastname: Wong
authors.forename: Yuk-Ching
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D059004:Topoisomerase I Inhibitors / Q000737:chemistry; D015010:Yersinia pestis / Q000201:enzymology / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D059004:Topoisomerase I Inhibitors; D001120:Arginine; D004264:DNA Topoisomerases, Type I