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Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking
Author(s) -
Cheng Bokun,
Zhou Qingxuan,
Weng Liwei,
Leszyk John D.,
Greenberg Marc M.,
TseDinh YukChing
Publication year - 2017
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.12517
Subject(s) - topoisomerase , dna , nuclease , escherichia coli , biochemistry , cleavage (geology) , chemistry , micrococcal nuclease , dna clamp , thymidine , biology , chromatin , gene , nucleosome , reverse transcriptase , rna , paleontology , fracture (geology)
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.