title: Residues of E. coli topoisomerase I conserved for interaction with a specific cytosine base to facilitate DNA cleavage.
abstract: Bacterial and archaeal topoisomerase I display selectivity for a cytosine base 4 nt upstream from the DNA cleavage site. Recently, the solved crystal structure of Escherichia coli topoisomerase I covalently linked to a single-stranded oligonucleotide revealed that R169 and R173 interact with the cytosine base at the -4 position via hydrogen bonds while the phenol ring of Y177 wedges between the bases at the -4 and the -5 position. Substituting R169 to alanine changed the selectivity of the enzyme for the base at the -4 position from a cytosine to an adenine. The R173A mutant displayed similar sequence selectivity as the wild-type enzyme, but weaker cleavage and relaxation activity. Mutation of Y177 to serine or alanine rendered the enzyme inactive. Although mutation of each of these residues led to different outcomes, R169, R173 and Y177 work together to interact with a cytosine base at the -4 position to facilitate DNA cleavage. These strictly conserved residues might act after initial substrate binding as a Molecular Ruler to form a protein-DNA complex with the scissile phosphate positioned at the active site for optimal DNA cleavage by the tyrosine hydroxyl nucleophile to facilitate DNA cleavage in the reaction pathway.
authors: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
authors: Gagandeep
authors: G
authors: Narula
authors: Yuk-Ching
authors: YC
authors: Tse-Dinh
mesh_terms: D019943:Amino Acid Substitution; D003596:Cytosine / Q000737:chemistry*; D053837:DNA Cleavage; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D003596:Cytosine; D004264:DNA Topoisomerases, Type I