PDBsum entry: 1i7d

Isomerase/DNA

PDB id: 1i7d

Contents

Protein chain

620 a.a. *

DNA/RNA

Ligands

SO4

Metals

_CL

Waters ×332

* Residue conservation analysis

PDB id:

1i7d

Name:

Isomerase/DNA

Title:

Noncovalent complex of e.Coli DNA topoisomerase iii with an 8-base single-stranded DNA oligonucleotide

Structure:

5'-d( Cp Gp Cp Ap Ap Cp Tp T)-3'. Chain: b. Engineered: yes. DNA topoisomerase iii. Chain: a. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Escherichia coli. Organism_taxid: 562. Strain: hms-83. Gene: topb. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Biol. unit:

Dimer (from PQS)

Resolution:

2.05Å R-factor: 0.233 R-free: 0.260

Authors:

A.Changela,R.J.Digate,A.Mondragon

Key ref:

A.Changela et al. (2001). Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. Nature, 411, 1077-1081. PubMed id: 11429611 DOI: 10.1038/35082615

Date:

08-Mar-01 Release date: 29-Jun-01

Protein chain

P14294  (TOP3_ECOLI) -  DNA topoisomerase 3

Seq: 653 a.a.

Struc: 620 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.5.99.1.2 - Dna topoisomerase.
• Reaction: ATP-independent breakage of single-stranded DNA, followed by passage and rejoining.

 Gene Ontology (GO) functional annotation 

• Cellular component: chromosome (1 term)
• Biological process: DNA metabolic process (3 terms)
• Biochemical function: nucleotide binding (8 terms)

DOI no: 10.1038/35082615

Nature 411:1077-1081 (2001)

PubMed id: 11429611

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

A.Changela, R.J.DiGate, A.Mondragón.

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 A 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.

Selected figure(s)

Figure 2.
Figure 2: Comparison of the bound ssDNA structure to B-DNA. a, Stereo view showing the superposition of the bound ssDNA molecule onto one strand of an ideal B-DNA model. Superposition of the two molecules using the six nucleotides at the 3' end of the oligonucleotide gives an r.m.s.d. value of 1.7 Å for all atoms. The bound ssDNA is depicted with grey bonds and coloured by atom, while the complementary strands of the B-DNA model are shown in green and blue. b, Stereo view of the modelling of B-DNA into the ssDNA binding groove. A hybrid B-DNA molecule was generated with the bound ssDNA molecule (coloured by atom) and a complementary strand from the B-DNA model (shown in blue). The faded regions of the complementary strand represent areas of the modelled DNA strand that would collide with the protein and prevent binding within the cleft.

Figure 4.
Figure 4: Active site and catalytic mechanism. a, Stereo view showing the E. coli DNA topoisomerase III active site in the presence and absence of DNA. Residues from the apo structure are shown in blue, while residues of the enzyme -DNA complex are coloured red. Secondary structural elements of domains I and III of the apo enzyme are depicted in light and dark shades of grey, respectively. Elements corresponding to domains I and III of the complex are coloured green and purple, respectively. b, A stereo view showing the active-site region formed at the interface of domains I and III in the presence of ssDNA. The bonds in the oligonucleotide are coloured yellow, and protein elements are coloured by domain as in Fig. 1a. Hydrogen bonding and electrostatic interactions are depicted by black dotted lines. A red dotted line highlights the orientation of Phe 328 towards the phosphate group of the putative scissile bond (green). c, Schematic diagram illustrating a possible mechanism of DNA cleavage used by type IA topoisomerases. A nearby group acting as a general base (B:) abstracts the proton from the tyrosine thus activating it for nucleophilic attack. In E. coli DNA topoisomerase III, Tyr 328 forms a covalent linkage with the 5' phosphate of the scissile bond while the pentavalent transition state is stabilized by Arg 330 and Lys 8. Cleavage of the ssDNA is facilitated by Glu 7 which acts as a general acid catalyst by donating a proton to the leaving 3'-oxygen atom of the scissile bond.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2001, 411, 1077-1081) copyright 2001.

Figures were selected by an automated process.