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Title: Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

Abstract

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNAmore » rearrangements.« less

Authors:
; ;  [1]
  1. (NWU)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1006354
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature; Journal Volume: 411; Journal Issue: 06, 2001
Country of Publication:
United States
Language:
ENGLISH
Subject:
36 MATERIALS SCIENCE; CATALYSIS; CHROMOSOMES; CLEAVAGE; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; DNA; DNA REPLICATION; ENZYMES; ESCHERICHIA COLI; FUNCTIONALS; MUTANTS; OLIGONUCLEOTIDES; PROTEINS; RESIDUES; RESOLUTION; TOPOLOGY; TRANSCRIPTION

Citation Formats

Changela, A., Digate, R.J., and Mondragon, A. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. United States: N. p., 2010. Web.
Changela, A., Digate, R.J., & Mondragon, A. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. United States.
Changela, A., Digate, R.J., and Mondragon, A. 2010. "Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule". United States. doi:.
@article{osti_1006354,
title = {Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule},
author = {Changela, A. and Digate, R.J. and Mondragon, A.},
abstractNote = {A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.},
doi = {},
journal = {Nature},
number = 06, 2001,
volume = 411,
place = {United States},
year = 2010,
month = 3
}
  • No abstract prepared.
  • In order to gain insights into the mechanism of ssDNA binding and recognition by Escherichia coli DNA topoisomerase I, the structure of the 67 kDa N-terminal fragment of topoisomerase I was solved in complex with ssDNA. The structure reveals a new conformational stage in the multistep catalytic cycle of type IA topoisomerases. In the structure, the ssDNA binding groove leading to the active site is occupied, but the active site is not fully formed. Large conformational changes are not seen; instead, a single helix parallel to the ssDNA binding groove shifts to clamp the ssDNA. The structure helps clarify themore » temporal sequence of conformational events, starting from an initial empty enzyme and proceeding to a ssDNA-occupied and catalytically competent active site.« less
  • Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIBmore » enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.« less
  • Type IB DNA topoisomerases are found in all eukarya, two families of eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria. They alter DNA topology by cleaving and resealing one strand of duplex DNA via a covalent DNA-(3-phosphotyrosyl)-enzyme intermediate. Bacterial type IB enzymes were discovered recently and are described as poxvirus-like with respect to their small size, primary structures, and bipartite domain organization. Here we report the 1.75-{angstrom} crystal structure of Deinococcus radiodurans topoisomerase IB (DraTopIB), a prototype of the bacterial clade. DraTopIB consists of an amino-terminal (N) {beta}-sheet domain (amino acids 1-90) and a predominantly {alpha}-helical carboxyl-terminal (C)more » domain (amino acids 91-346) that closely resemble the corresponding domains of vaccinia virus topoisomerase IB. The five amino acids of DraTopIB that comprise the catalytic pentad (Arg-137, Lys-174, Arg-239, Asn-280, and Tyr-289) are preassembled into the active site in the absence of DNA in a manner nearly identical to the pentad configuration in human topoisomerase I bound to DNA. This contrasts with the apoenzyme of vaccinia topoisomerase, in which three of the active site constituents are either displaced or disordered. The N and C domains of DraTopIB are splayed apart in an 'open' conformation, in which the surface of the catalytic domain containing the active site is exposed for DNA binding. A comparison with the human topoisomerase I-DNA cocrystal structure suggests how viral and bacterial topoisomerase IB enzymes might bind DNA circumferentially via movement of the N domain into the major groove and clamping of a disordered loop of the C domain around the helix.« less