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 t

SUBMITTER: Cheng B 

PROVIDER: S-EPMC5235945 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature