Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking

Bokun Cheng, Qingxuan Zhou, Liwei Weng, John D. Leszyk, Marc M. Greenberg, Yuk Ching Tse-Dinh

Research output: Contribution to journalLetter

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.

Original languageEnglish (US)
Pages (from-to)28-38
Number of pages11
JournalFEBS Letters
Volume591
Issue number1
DOIs
StatePublished - Jan 1 2017
Externally publishedYes

Fingerprint

Type I DNA Topoisomerase
Crosslinking
Escherichia coli
DNA
Thymidine
Substrates
Oxidants
Trypsin
Lysine
Mass spectrometry
Digestion
Mass Spectrometry
Topology
Enzymes
Proteins

Keywords

  • crosslinking
  • DNA–protein interactions
  • topoisomerase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking. / Cheng, Bokun; Zhou, Qingxuan; Weng, Liwei; Leszyk, John D.; Greenberg, Marc M.; Tse-Dinh, Yuk Ching.

In: FEBS Letters, Vol. 591, No. 1, 01.01.2017, p. 28-38.

Research output: Contribution to journalLetter

Cheng, Bokun ; Zhou, Qingxuan ; Weng, Liwei ; Leszyk, John D. ; Greenberg, Marc M. ; Tse-Dinh, Yuk Ching. / Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking. In: FEBS Letters. 2017 ; Vol. 591, No. 1. pp. 28-38.
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