DNA helicase

 

Recombination in bacteria requires the isomerisation of the Holliday junction in a process revolving around the activities of three enzymes each with specific functions. RuvB, the second of the three enzymes involved, catalyses the migration of the branches of DNA before the cleavage step occurs, so that the cleavage and rejoining can produce recombinant products. It works by using ATP hydrolysis to power conformational change in the DNA binding domain, restructuring the Holliday junction. The ATP binding domain shows homology to the AAA family (ATP associated activities) in that it has a Rossman fold and both Walker A and Walker B motifs. Here, the enzyme from Thermus thermophilus is analysed; it displays significant sequence identity with the E.coli enzyme and both share similar active sites and probable catalytic mechanisms, as well as the same overall fold.

 

Reference Protein and Structure

Sequence
Q5SL87 UniProt (3.6.4.12) IPR004605 (Sequence Homologues) (PDB Homologues)
Biological species
Thermus thermophilus HB8 (Bacteria) Uniprot
PDB
1hqc - STRUCTURE OF RUVB FROM THERMUS THERMOPHILUS HB8 (3.2 Å) PDBe PDBsum 1hqc
Catalytic CATH Domains
1.10.8.60 CATHdb 3.40.50.300 CATHdb (see all for 1hqc)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.6.4.12)

ATP(4-)
CHEBI:30616ChEBI
+
water
CHEBI:15377ChEBI
→
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: 3' to 5' DNA helicase, 3'-5' DNA helicase, 3'-5' PfDH, 5' to 3' DNA helicase, AvDH1, BACH1 helicase, BcMCM, BLM protein, BRCA1-associated C-terminal helicase, CeWRN-1, Dbp9p, DmRECQ5, DNA helicase 120, DNA helicase A, DNA helicase E, DNA helicase II, DNA helicase III, DNA helicase RECQL5', DNA helicase VI, DnaB, DnaB helicase E1, Helicase HDH IV, Hel E, Helicase DnaB, Helicase domain of bacteriophage T7 gene 4 protein helicase, PcrA helicase, UvrD, HHcsA, Hmi1p, HPif1, MCM helicase, MCM protein, MER3 helicase, MER3 protein, MPH1, PcrA, PDH120, PfDH A, Pfh1p, PIF1,

Enzyme Mechanism

Introduction

ATP hydrolysis occurs through nucleophilic attack by water on the electrophilic phosphorous atom of the gamma phosphate of ATP to produce a pentavalent phosphate transition state, stabilised by contacts with Mg2+, Arg 205, Lys 51 and Thr 146. Collapse of this intermediate releases ADP and Pi, and is used to drive the conversion of the inactive homoheptamer to a homohexameric structure, thus causing branch migration of the DNA.

Catalytic Residues Roles

UniProt PDB* (1hqc)
Thr52, Asp97 Thr52A, Asp97A Form Mg2+ binding site
Lys51, Thr146 Lys51A, Thr146A Acts to stabilise the pentavalent phosphate transition state that forms during the reaction through electrostatic contacts with the beta phosphate.
Arg205 Arg205A Stabilises the pentavalent phosphate transition state through electrostatic contacts with the gamma phosphate of ATP. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

References

  1. Yamada K et al. (2001), Proc Natl Acad Sci U S A, 98, 1442-1447. Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8. DOI:10.1073/pnas.031470598. PMID:11171970.
  2. Caruthers JM et al. (2002), Curr Opin Struct Biol, 12, 123-133. Helicase structure and mechanism. DOI:10.1016/S0959-440X(02)00298-1.
  3. Yamada K et al. (2002), Mol Cell, 10, 671-681. Crystal Structure of the RuvA-RuvB Complex. DOI:10.1016/S1097-2765(02)00641-X.
  4. Putnam CD et al. (2001), J Mol Biol, 311, 297-310. Structure and mechanism of the RuvB Holliday junction branch migration motor. DOI:10.1006/jmbi.2001.4852. PMID:11478862.
  5. Hishida T et al. (1999), J Biol Chem, 274, 25335-25342. Role of Walker Motif A of RuvB Protein in Promoting Branch Migration of Holliday Junctions. WALKER MOTIF A MUTATIONS AFFECT ATP BINDING, ATP HYDROLYZING, AND DNA BINDING ACTIVITIES OF RuvB. DOI:10.1074/jbc.274.36.25335. PMID:10464259.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Thr146A electrostatic stabiliser
Arg205A electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Peter Sarkies, Gemma L. Holliday, Charity Hornby