DNA polymerase eta

 

DNA polymerase eta is a translesion synthesis (TLS) polymerase. The TLS polymerases are an important part of the DNA synthesis pathway which aid in resistance to DNA damage. They are crucial in overcoming DNA damage in cells by bypassing DNA damage lesions during DNA replication, which may otherwise stall the replication fork and lead to cell death. Eta polymerases are of interest because when mutated, can lead to cancer and are implicated as causes of antibiotic and chemotherapy resistance.

 

Reference Protein and Structure

Sequence
Q9Y253 UniProt (2.7.7.7) IPR043502, IPR036775 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
4ecs - Human DNA polymerase eta - DNA ternary complex: Reaction in the AT crystal at pH 7.0 for 80 sec (1.951 Å) PDBe PDBsum 4ecs
Catalytic CATH Domains
3.30.70.270 CATHdb 2.30.40.20 CATHdb (see all for 4ecs)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.7)

2'-deoxyribonucleoside 5'-triphosphate(4-)
CHEBI:61560ChEBI
+
DNA polyanion
CHEBI:83828ChEBI
→
diphosphate(3-)
CHEBI:33019ChEBI
+
DNA Polymer
CHEBI:X00680X00680
Alternative enzyme names: DNA duplicase, DNA nucleotidyltransferase, DNA nucleotidyltransferase (DNA-directed), DNA polymerase, DNA polymerase alpha, DNA polymerase beta, DNA polymerase gamma, DNA polymerase I, DNA polymerase II, DNA polymerase III, DNA replicase, DNA-dependent DNA polymerase, Klenow fragment, Taq DNA polymerase, Taq Pol I, Tca DNA polymerase, Deoxynucleate polymerase, Deoxyribonucleate nucleotidyltransferase, Deoxyribonucleic acid duplicase, Deoxyribonucleic acid polymerase, Deoxyribonucleic duplicase, Deoxyribonucleic polymerase, Deoxyribonucleic polymerase I, Duplicase, Sequenase,

Enzyme Mechanism

Introduction

Glu116 deprotonates the 3'-OH of the DNA substrate, activating it to bind to the α-phosphate of the nucleotide. This causes the phospho-anhydride bond to break with the diphosphate leaving group. This reaction is energetically and structurally the most favourable of all the alternative mechanisms.

Catalytic Residues Roles

UniProt PDB* (4ecs)
Glu116 Glu116(119)A Glu116 deprotonates the 3'OH group on the DNA chain, activating it to perform a nucleophilic attack on the phosphate of the substrate. metal ligand, proton acceptor
Arg61, Arg55, Tyr52, Lys231 Arg61(64)A, Arg55(58)A, Tyr52(55)A, Lys231(234)A Residues Tyr52, Arg55, Arg61 and Lys231 stabilise intermediates formed with the substrate by forming hydrogen bonds with the substrate and electrostatically stabilising the negative charge on the phosphate groups. electrostatic stabiliser
Ser113, Asp13, Asp115, Met14 (main-C) Ser113(116)A, Asp13(16)A, Asp115(118)A, Met14(17)A (main-C) Residues Asp13, Met14(main-N), Ser113, Asp115 and Glu116 coordinate metal ions to the active site. metal ligand
*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, overall reactant used, bimolecular nucleophilic substitution, overall product formed

References

  1. Wilson KA et al. (2019), ACS Catal, 9, 2543-2551. Exploring the Identity of the General Base for a DNA Polymerase Catalyzed Reaction Using QM/MM: The Case Study of Human Translesion Synthesis Polymerase η. DOI:10.1021/acscatal.8b04889.
  2. Biertümpfel C et al. (2010), Nature, 465, 1044-1048. Structure and mechanism of human DNA polymerase eta. DOI:10.1038/nature09196. PMID:20577208.

Step 1. Glu116 deprotonates the 3'-OH of DNA.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp115(118)A metal ligand
Met14(17)A (main-C) metal ligand
Glu116(119)A metal ligand
Ser113(116)A metal ligand
Asp13(16)A metal ligand
Arg55(58)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Lys231(234)A electrostatic stabiliser
Glu116(119)A proton acceptor
Arg61(64)A electrostatic stabiliser

Chemical Components

proton transfer

Step 2. The deprotonated 3' deoxyribose attacks the α-phosphate of the nucleotide, breaking the phospho-anhydride bond with the diphosphate leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys231(234)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Arg55(58)A electrostatic stabiliser
Asp13(16)A metal ligand
Met14(17)A (main-C) metal ligand
Asp115(118)A metal ligand
Glu116(119)A metal ligand
Ser113(116)A metal ligand
Arg61(64)A electrostatic stabiliser

Chemical Components

overall reactant used, ingold: bimolecular nucleophilic substitution, overall product formed
Download: Image, Marvin File

Step 1. Glu116 deprotonates the 3'-OH of DNA.

Step 2. The deprotonated 3' deoxyribose attacks the α-phosphate of the nucleotide, breaking the phospho-anhydride bond with the diphosphate leaving group.

Products.

Introduction

This is the "WMSA" mechanism, in which two Mg2+ ions coordinate two water molecules near the dATP and DNA chain. Firstly, a water molecule deprotonates the 3'-OH group of the main DNA chain, which then protonates the distal phosphate of the dATP nucleotide. This causes the 3' oxygen to attack the nucleotide α-phosphate. The diphosphate group leaves following proton transfer from the distal γ-phosphate to the β-phosphate. This mechanism was, however, marked as a unlikely mechanism because of the large energy needed to be performed.

Catalytic Residues Roles

UniProt PDB* (4ecs)
Arg55 Arg55(58)A Arg55 stabilizes the phosphate structure on the dATP group with hydrogen bonds towards the phosphate oxygens. electrostatic stabiliser
Tyr52 Tyr52(55)A Tyr52 stabilizes the phosphate structure on the dATP group with hydrogen bonds towards the phosphate oxygens. electrostatic stabiliser
Glu116 Glu116(119)A Glu116 coordinates the metal ions. metal ligand
Ser113 Ser113(116)A Ser113 coordinates the metal ions. metal ligand
Lys231 Lys231(234)A Lys231 stabilizes the phosphate structure on the dATP group with hydrogen bonds towards the phosphate oxygens. electrostatic stabiliser
Asp13 Asp13(16)A Asp13 coordinates the metal ions. metal ligand
Asp115 Asp115(118)A Asp115 coordinates the metal ions. metal ligand
Met14 (main-C) Met14(17)A (main-C) Met14 (main chain) coordinates the metal ions. metal ligand
*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, overall reactant used, overall product formed

References

  1. Wilson KA et al. (2019), ACS Catal, 9, 2543-2551. Exploring the Identity of the General Base for a DNA Polymerase Catalyzed Reaction Using QM/MM: The Case Study of Human Translesion Synthesis Polymerase η. DOI:10.1021/acscatal.8b04889.
  2. Stevens DR et al. (2018), J Am Chem Soc, 140, 8965-8969. Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations. DOI:https://doi.org/10.1021/jacs.8b05177.

Step 1. The Mg2+ ions coordinate two water molecules near the dATP and DNA chain which cause a proton relay that transfers a proton from the DNA deoxyribose to a water molecule and from the water molecule to a γ-phosphate on the dATP. Asp115, Met14, Glu116, Ser113 and Asp13 coordinate the 2 Mg2+ ions while Tyr52, Lys231 and Arg55 stabilize the negatively charged phosphate groups on the dATP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys231(234)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Arg55(58)A electrostatic stabiliser
Asp13(16)A metal ligand
Ser113(116)A metal ligand
Glu116(119)A metal ligand
Met14(17)A (main-C) metal ligand
Asp115(118)A metal ligand

Chemical Components

proton transfer

Step 2. The 3' OH group of the DNA performs a nucleophilic attack on the α-phosphate of the dATP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser113(116)A metal ligand
Glu116(119)A metal ligand
Asp115(118)A metal ligand
Met14(17)A (main-C) metal ligand
Asp13(16)A metal ligand
Arg55(58)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Lys231(234)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used

Step 3. A proton donated in the first step is shuffled to the β-phosphate and the bond between the α and β phosphate is broken.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser113(116)A metal ligand
Glu116(119)A metal ligand
Asp115(118)A metal ligand
Met14(17)A (main-C) metal ligand
Asp13(16)A metal ligand
Arg55(58)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Lys231(234)A electrostatic stabiliser

Chemical Components

proton transfer, overall product formed
Download: Image, Marvin File

Step 1. The Mg2+ ions coordinate two water molecules near the dATP and DNA chain which cause a proton relay that transfers a proton from the DNA deoxyribose to a water molecule and from the water molecule to a γ-phosphate on the dATP. Asp115, Met14, Glu116, Ser113 and Asp13 coordinate the 2 Mg2+ ions while Tyr52, Lys231 and Arg55 stabilize the negatively charged phosphate groups on the dATP.

Step 2. The 3' OH group of the DNA performs a nucleophilic attack on the α-phosphate of the dATP.

Step 3. A proton donated in the first step is shuffled to the β-phosphate and the bond between the α and β phosphate is broken.

Products.

Introduction

Glu116 deprotonates Ser113 which then proceeds to deprotonate the 3'-OH of the DNA chain deoxyribose. This leads to the negatively charged oxygen of the DNA chain binding to the α-phosphate of the nucleotide, detaching the nucleotide from two phosphates. However, this mechanism is disputed because its not oriented correctly in crystal structures. Also, mutagenesis studies show the enzyme retains 95% of the kcat value in the S113A mutant.

Catalytic Residues Roles

UniProt PDB* (4ecs)
Glu116 Glu116(119)A Glu116 acts as a proton acceptor for Ser113 and ti also coordinates a Mg2+ ion. metal ligand, proton acceptor
Ser113 Ser113(116)A Ser113 acts as a proton acceptor for the 3'OH group and ti also coordinates a Mg2+ ion. metal ligand, proton acceptor
Arg55, Tyr52, Lys231 Arg55(58)A, Tyr52(55)A, Lys231(234)A Coordinates and stabilizes the phosphate groups of the nucleotide. electrostatic stabiliser
Asp13, Asp115, Met14 (main-C) Asp13(16)A, Asp115(118)A, Met14(17)A (main-C) Coordinates the Mg2+ ion. metal ligand
*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, overall product formed, overall reactant used

References

  1. Wilson KA et al. (2019), ACS Catal, 9, 2543-2551. Exploring the Identity of the General Base for a DNA Polymerase Catalyzed Reaction Using QM/MM: The Case Study of Human Translesion Synthesis Polymerase η. DOI:10.1021/acscatal.8b04889.
  2. Nakamura T et al. (2012), Nature, 487, 196-201. Watching DNA polymerase η make a phosphodiester bond. DOI:10.1038/nature11181. PMID:22785315.

Step 1. Glu116 deprotonates Ser113 and Ser113, consequently deprotonating the 3'-OH from the main DNA chain.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp115(118)A metal ligand
Met14(17)A (main-C) metal ligand
Glu116(119)A metal ligand
Ser113(116)A metal ligand
Asp13(16)A metal ligand
Arg55(58)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Lys231(234)A electrostatic stabiliser
Ser113(116)A proton acceptor
Glu116(119)A proton acceptor

Chemical Components

proton transfer

Step 2. The deprotonated 3' deoxyribose attacks the α-phosphate of the nucleotide, breaking the phospho-anhydride bond with the diphosphate leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys231(234)A electrostatic stabiliser
Tyr52(55)A electrostatic stabiliser
Arg55(58)A electrostatic stabiliser
Asp13(16)A metal ligand
Ser113(116)A metal ligand
Glu116(119)A metal ligand
Met14(17)A (main-C) metal ligand
Asp115(118)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, overall product formed, overall reactant used
Download: Image, Marvin File

Step 1. Glu116 deprotonates Ser113 and Ser113, consequently deprotonating the 3'-OH from the main DNA chain.

Step 2. The deprotonated 3' deoxyribose attacks the α-phosphate of the nucleotide, breaking the phospho-anhydride bond with the diphosphate leaving group.

Products.

Contributors

Marko Babić, Noa Marson