title: Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking.
abstract: Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, USA.
authors.forename: Bokun
authors.initials: B
authors.lastname: Cheng
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.|Biomolecular Sciences Institute, Florida International University, Miami, FL, USA.
authors.forename: Qingxuan
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Liwei
authors.initials: L
authors.lastname: Weng
authors.affiliation: Department of Biochemistry and Molecular Pharmacology and Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, MA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Leszyk
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Marc M
authors.initials: MM
authors.lastname: Greenberg
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.|Biomolecular Sciences Institute, Florida International University, Miami, FL, USA.
authors.forename: Yuk-Ching
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D008239:Lysine / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D009841:Oligonucleotides / Q000378:metabolism; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D009841:Oligonucleotides; D010455:Peptides; D008836:Micrococcal Nuclease; D004264:DNA Topoisomerases, Type I; D008239:Lysine
keywords: DNA-protein interactions; crosslinking; topoisomerase