Structural Studies of E. coli Topoisomerase III-DNA Complexes Reveal a Novel Type IA Topoisomerase-DNA Conformational Intermediate

Anita Changela, Russell J. DiGate, Alfonso Mondragón*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

34 Scopus citations

Abstract

Escherichia coli DNA topoisomerase III belongs to the type IA family of DNA topoisomerases, which transiently cleave single-stranded DNA (ssDNA) via a 5′ phosphotyrosine intermediate. We have solved crystal structures of wild-type E. coli topoisomerase III bound to an eight-base ssDNA molecule in three different pH environments. The structures reveal the enzyme in three distinct conformational states while bound to DNA. One conformation resembles the one observed previously with a DNA-bound, catalytically inactive mutant of topoisomerase III where DNA binding realigns catalytic residues to form a functional active site. Another conformation represents a novel intermediate in which DNA is bound along the ssDNA-binding groove but does not enter the active site, which remains in a catalytically inactive, closed state. A third conformation shows an intermediate state where the enzyme is still in a closed state, but the ssDNA is starting to invade the active site. For the first time, the active site region in the presence of both the catalytic tyrosine and ssDNA substrate is revealed for a type IA DNA topoisomerase, although there is no evidence of ssDNA cleavage. Comparative analysis of the various conformational states suggests a sequence of domain movements undertaken by the enzyme upon substrate binding.

Original languageEnglish (US)
Pages (from-to)105-118
Number of pages14
JournalJournal of Molecular Biology
Volume368
Issue number1
DOIs
StatePublished - Apr 20 2007

Funding

We thank A. Patel for technical assistance and Michael Blum for assistance with using the MARMosaic detector. Research was supported by NIH GM51350 to A.M. and an NRSA Institutional Training Grant in Molecular Biophysics (NIH GM08382) to A.C. We acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University. Support from the R.H. Lurie Cancer Center of Northwestern University to the Structural Biology Center is acknowledged. Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center at the Advanced Photon Source (APS) and at the Stanford Synchrotron Radiation Laboratory (SSRL). DND-CAT is supported by DuPont, Dow, and the NSF and use of the APS is supported by the DOE. SSRL is operated by the DOE, Office of Basic Energy Sciences. The SSRL Biotechnology Program is supported by the NIH and the DOE.

Keywords

  • complexes
  • ssDNA
  • structure
  • topoisomerase III

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology

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