title: Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I.
abstract: DNA topoisomerases control DNA topology by breaking and rejoining DNA strands via covalent complexes with cleaved DNA substrate as catalytic intermediates. Here we report the structure of Escherichia coli topoisomerase I catalytic domain (residues 2-695) in covalent complex with a cleaved single-stranded oligonucleotide substrate, refined to 2.3-Å resolution. The enzyme-substrate intermediate formed after strand cleavage was captured due to the presence of the D111N mutation. This structure of the covalent topoisomerase-DNA intermediate, previously elusive for type IA topoisomerases, shows distinct conformational changes from the structure of the enzyme without bound DNA and provides detailed understanding of the covalent catalysis required for strand cleavage to take place. The portion of cleaved DNA 5' to the site of cleavage is anchored tightly with extensive noncovalent protein-DNA interactions as predicted by the "enzyme-bridged" model. Distortion of the scissile strand at the -4 position 5' to the cleavage site allows specific selectivity of a cytosine base in the binding pocket. Many antibacterial and anticancer drugs initiate cell killing by trapping the covalent complexes formed by topoisomerases. We have demonstrated in previous mutagenesis studies that accumulation of the covalent complex of bacterial topoisomerase I is bactericidal. This structure of the covalent intermediate provides the basis for the design of novel antibiotics that can trap the enzyme after formation of the covalent complex.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York, NY 10595, USA.
authors.forename: Zhongtao
authors.initials: Z
authors.lastname: Zhang
authors.forename: Bokun
authors.initials: B
authors.lastname: Cheng
authors.forename: Yuk-Ching
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; 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; D017434:Protein Structure, Tertiary
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004264:DNA Topoisomerases, Type I