A catalytic domain of eukaryotic DNA topoisomerase I

Chonghui Cheng*, Stewart Shuman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314- amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT ↓. Here we show that Topo-(81- 314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single- turnover DNA cleavage by 10-3.6, but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a fall-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.

Original languageEnglish (US)
Pages (from-to)11589-11595
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number19
DOIs
StatePublished - May 8 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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