DNA topoisomerase (type IB)

 

DNA topoisomerase IB (TopIB) is an enzyme that acts to relax both negative and positive supercoils generated during transcription and DNA replication. Due to of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. TopIB mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723 in human, Tyr-274 in the viral protein) at a DNA phosphodiester bond forming a 3-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3-phosphotyrosyl TopIB DNA complex remains low. A variety of DNA lesions and drugs have been shown to stabilize the covalent 3-phosphotyrosyl intermediate.

Unlike most TopIBs, the enzyme isolated from the vaccinia virus exhibits specificity for cleavage at a consensus sequence: 5'-(T/C)CCTT-3' in the scissile strand with cleavage occuring after the last base. The viral TopIB is also the smallest topoisomerase known and is unusual in that it is resistant to the potent chemotherapeutic agent camptothecin (CPT). CPT is a natural product that was originally discovered because of its antitumor activity and was later demonstrated to cause the accumulation of TopIB DNA adducts in vitro and in vivo. CPTs bind the covalent 3-phosphotyrosyl intermediate and specifically block DNA religation, thus converting TopIB into a DNA-damaging agent. TopIB is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific. During DNA replication, the replication fork is thought to collide with the trapped TopIB DNA complexes, resulting in double-strand breaks and ultimately cell death.

 

Reference Protein and Structure

Sequence
P68698 UniProt (5.6.2.1) IPR001631 (Sequence Homologues) (PDB Homologues)
Biological species
Vaccinia virus WR (Virus) Uniprot
PDB
1a41 - TYPE 1-TOPOISOMERASE CATALYTIC FRAGMENT FROM VACCINIA VIRUS (2.3 Å) PDBe PDBsum 1a41
Catalytic CATH Domains
3.90.15.10 CATHdb 1.20.120.380 CATHdb (see all for 1a41)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:5.6.2.1)

double-stranded DNA
CHEBI:4705ChEBI
→
double-stranded DNA
CHEBI:4705ChEBI
Alternative enzyme names: Omega-protein, Nicking-closing enzyme, Relaxing enzyme, Swivelase, Type I DNA topoisomerase, Untwisting enzyme, DNA topoisomerase I,

Enzyme Mechanism

Introduction

Tyr274 is the nucleophile for in-line displacement at the DNA phosphate group with the 5'-hydroxyl as the leaving group. The transition state is stabilised by Arg130 binding to one non-bridging oxygen, Arg223 and His265 binding to the other non-bridging oxygen, and Arg223 and Lys167 binding to the 5'-hydroxyl leaving group. Either Arg223 or a water molecule is involved in general base catalysis with respect to Tyr274 while Lys167 and Arg130 are involved in general acid catalysis with respect to the 5'-hydroxyl leaving group. Rotation of the DNA-enzyme intermediate around the uncleaved strand then occurs, relaxing the supercoiled DNA. The 5'-hydroxyl is then the nucleophile for in-line displacement at the DNA-enzyme intermediate. The transition state is stabilised in a similar way to that of DNA cleavage. Tyr274 is the leaving group and is protonated by Arg223/water.

Catalytic Residues Roles

UniProt PDB* (1a41)
Lys167, Arg130 Lys167(87)A, Arg130(50)A Lys167 coordinates the 5'-hydroxyl leaving group during DNA cleavage and is thought to be involved in general acid catalysis. Although the exact mechanism of protonation is uncertain, there is evidence to suggest that Arg130 and Lys167 act in a concerted fashion in the protonation of the O-5'. attractive charge-charge interaction, hydrogen bond donor, proton acceptor, proton donor, proton relay, electrostatic stabiliser
Tyr274 Tyr274(194)A Tyr274 is the nucleophile for attack on the phosphate group during DNA cleavage. The pKa of the phenol is thought to be lowered by Arg223 to make it a better nucleophile. Arg223 may also act as a base to deprotonate Tyr274, or a water molecule could fill this role. Tyr274 is the leaving group during DNA ligation and is protonated by Arg223/water. hydrogen bond acceptor, hydrogen bond donor, nucleophile, proton acceptor, proton donor, nucleofuge
Arg223 Arg223(143)A Arg223 stabilises the transition state of DNA cleavage by coordinating one of the phosphate non-bridging oxygens. It is though to lower the pKa of Tyr274 and may be involved in the deprotonation of Tyr274 during cleavage. If the latter is the case then it would presumably act as an acid during DNA ligation, as well as stabilising the transition state. attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
His265 His265(185)A His265 stabilises the transition states of DNA cleavage and ligation by coordinating to one of the phosphate non-bridging oxygens. hydrogen bond donor, 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

proton transfer, bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton relay, rate-determining step, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated

References

  1. Wittschieben J et al. (1997), Nucleic Acids Res, 25, 3001-3008. Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167. DOI:10.1093/nar/25.15.3001. PMID:9224599.
  2. Yakovleva L et al. (2008), J Biol Chem, 283, 16093-16103. Chemical and Traditional Mutagenesis of Vaccinia DNA Topoisomerase Provides Insights to Cleavage Site Recognition and Transesterification Chemistry. DOI:10.1074/jbc.m801595200. PMID:18367446.
  3. Perry K et al. (2006), Mol Cell, 23, 343-354. Structural Basis for Specificity in the Poxvirus Topoisomerase. DOI:10.1016/j.molcel.2006.06.015. PMID:16885024.
  4. Davies DR et al. (2006), J Mol Biol, 357, 1202-1210. The Structure of the Transition State of the Heterodimeric Topoisomerase I of Leishmania donovani as a Vanadate Complex with Nicked DNA. DOI:10.1016/j.jmb.2006.01.022. PMID:16487540.
  5. Interthal H et al. (2004), J Biol Chem, 279, 2984-2992. The Role of Lysine 532 in the Catalytic Mechanism of Human Topoisomerase I. DOI:10.1074/jbc.m309959200. PMID:14594810.
  6. Krogh BO et al. (2002), J Biol Chem, 277, 5711-5714. Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB. DOI:10.1074/jbc.c100681200. PMID:11756402.
  7. Stivers JT et al. (2000), Biochemistry, 39, 5561-5572. Stereochemical Outcome and Kinetic Effects ofRp- andSp-Phosphorothioate Substitutions at the Cleavage Site of Vaccinia Type I DNA Topoisomerase†. DOI:10.1021/bi992429c. PMID:10820030.
  8. Cheng C et al. (1998), Cell, 92, 841-850. Conservation of Structure and Mechanism between Eukaryotic Topoisomerase I and Site-Specific Recombinases. DOI:10.1016/s0092-8674(00)81411-7. PMID:9529259.
  9. Stewart L et al. (1998), Science, 279, 1534-1541. A Model for the Mechanism of Human Topoisomerase I. DOI:10.1126/science.279.5356.1534. PMID:9488652.
  10. Cheng C et al. (1997), J Biol Chem, 272, 8263-8269. Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis. DOI:10.1074/jbc.272.13.8263. PMID:9079646.
  11. Shuman S et al. (1989), Proc Natl Acad Sci U S A, 86, 9793-9797. Mapping the active-site tyrosine of vaccinia virus DNA topoisomerase I. DOI:10.1073/pnas.86.24.9793. PMID:2557629.

Step 1. Water deprotonates Tyr274, initiating a nucleophilic in-line substitution at the DNA phosphate with a trigonal bipyramidal transition state. The leaving 5'-hydroxyl is initially protonated by Lys167, which in turn deprotonates Arg130, which deprotonates the initial acidic water in a concerted fashion.

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Catalytic Residues Roles

Residue Roles
Tyr274(194)A hydrogen bond donor
Lys167(87)A proton relay, hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Arg130(50)A hydrogen bond donor, proton relay, electrostatic stabiliser, attractive charge-charge interaction
His265(185)A hydrogen bond donor, electrostatic stabiliser
Arg223(143)A hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Lys167(87)A proton acceptor
Arg130(50)A proton donor, proton acceptor
Tyr274(194)A nucleophile, proton donor
Lys167(87)A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, enzyme-substrate complex formation, intermediate formation, overall reactant used, proton relay, rate-determining step

Step 2. Before this catalytic step occurs the DNA-enzyme intermediate rotates around the uncleaved DNA strand, relaxing the supercoiled DNA. Water deprotonates Arg130, which in turn deprotonates Lys167, which deprotonates the rotated 5'-OH initiating a nucleophilic in-line substitution at the DNA phosphate with a trigonal bipyramidal transition state/ The eliminated Tyr274 deprotonates the initial acidic water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr274(194)A hydrogen bond acceptor
Lys167(87)A proton relay, hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Arg130(50)A hydrogen bond donor, proton relay, electrostatic stabiliser, attractive charge-charge interaction
His265(185)A hydrogen bond donor, electrostatic stabiliser
Arg223(143)A hydrogen bond donor, electrostatic stabiliser, attractive charge-charge interaction
Lys167(87)A proton acceptor
Tyr274(194)A proton acceptor
Arg130(50)A proton donor, proton acceptor
Tyr274(194)A nucleofuge
Lys167(87)A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated, proton relay
Download: Image, Marvin File

Step 1. Water deprotonates Tyr274, initiating a nucleophilic in-line substitution at the DNA phosphate with a trigonal bipyramidal transition state. The leaving 5'-hydroxyl is initially protonated by Lys167, which in turn deprotonates Arg130, which deprotonates the initial acidic water in a concerted fashion.

Step 2. Before this catalytic step occurs the DNA-enzyme intermediate rotates around the uncleaved DNA strand, relaxing the supercoiled DNA. Water deprotonates Arg130, which in turn deprotonates Lys167, which deprotonates the rotated 5'-OH initiating a nucleophilic in-line substitution at the DNA phosphate with a trigonal bipyramidal transition state/ The eliminated Tyr274 deprotonates the initial acidic water molecule.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, Anna Waters, Craig Porter