8-oxoguanine DNA-glycosylase (ExoA family)

 

Human apurinic/apyrimidinic endonuclease I (HAP1) sourced from Homo sapiens is an essential DNA repair enzyme that initiates the removal of apurinic/apyrimidinic sites from DNA, excises 3' replication-blocking moieties and modulates DNA binding activity of several transcriptional regulators. HAP1 recognises abasic sites in ds DNA and makes a single nick in the backbone 5' to the abasic site generating the 3' hydroxyl required by the repair polymerase. Abasic lesions arise at high frequency in DNA due to spontaneous, mutagen induced or glycosylase-mediated hydrolysis of the N-glycosylic bond and represent cytotoxic and mutagenic lesions if uncorrected. HAP1 also exhibits 3'phosphodiesterase, 3'->5' exonuclease and RNase H activity. The Ref-1 (redox effector factor) activity of HAP1 is to regulate the DNA-binding affinity of transcription factors such as Jun/Fos and NF-kB through a redox mechanism.

 

Reference Protein and Structure

Sequence
P27695 UniProt (3.1.-.-) IPR004808 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1bix - THE CRYSTAL STRUCTURE OF THE HUMAN DNA REPAIR ENDONUCLEASE HAP1 SUGGESTS THE RECOGNITION OF EXTRA-HELICAL DEOXYRIBOSE AT DNA ABASIC SITES (2.2 Å) PDBe PDBsum 1bix
Catalytic CATH Domains
3.60.10.10 CATHdb (see all for 1bix)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:4.2.99.18)

water
CHEBI:15377ChEBI
+
DNA 8-oxoguanine
CHEBI:137052ChEBI
→
DNA with 3-terminal trans-a,b-unsaturated sugar
CHEBI:137051ChEBI
+
DNA 5'-phosphate
CHEBI:4294ChEBI
+
hydron
CHEBI:15378ChEBI
+
7,8-dihydro-8-oxoguanine
CHEBI:52617ChEBI
Alternative enzyme names: E. coli endonuclease III, Micrococcus luteus UV endonuclease, AP endonuclease class I, AP lyase, AP site-DNA 5'-phosphomonoester-lyase, X-ray endonuclease III, Deoxyribonuclease (apurinic or apyrimidinic), Endodeoxyribonuclease (apurinic or apyrimidinic), Phage-T(4) UV endonuclease, Phage-T4 UV endonuclease, E.coli endonuclease III,

Enzyme Mechanism

Introduction

Asp210 acts as a general base activating the water molecule for nucleophilic attack on the scissile phosphate. Asn212 positions the water for nucleophilic attack in a hydrogen bond network with Asp210. The pentaphosphate intermediate formed is stabilized by His309, Asp283, the Mg ions and Tyr171. 7,8-dihydro-8-oxoguanine is then cleaved from the phosphate, this cleavage is catalyzed through electrostatic stabilization.

Catalytic Residues Roles

UniProt PDB* (1bix)
Glu96, Asp308, Asp70 Glu96(65)A, Asp308(277)A, Asp70(39)A Involved predominately in metal binding. metal ligand
Asp283 Asp283(252)A Asp 283 stabilises His 309 through hydrogen bonding. electrostatic stabiliser
His309 His309(278)A His 309 contributes to polarisation of the DNA phosphate group. His 309 also stabilises the transition state. metal ligand, electrostatic stabiliser
Tyr171 Tyr171(140)A stabilizes the phosphate intermediate metal ligand, electrostatic stabiliser
Asp210 Asp210(179)A Asp 210 acts as a general base, deprotonating a water molecule which attacks the scissile phosphate. increase nucleophilicity, metal ligand, proton acceptor
*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 addition, overall reactant used, intramolecular elimination, overall product formed

References

  1. Batebi H et al. (2018), Proteins, 86, 439-453. Role of AP-endonuclease (Ape1) active site residues in stabilization of the reactant enzyme-DNA complex. DOI:10.1002/prot.25460. PMID:29344998.
  2. Borjigin M et al. (2012), FEBS Lett, 586, 242-247. Chinese hamster AP endonuclease operates by a two-metal ion assisted catalytic mechanism. DOI:10.1016/j.febslet.2011.12.025. PMID:22209979.
  3. Mundle ST et al. (2004), DNA Repair (Amst), 3, 1447-1455. Novel role of tyrosine in catalysis by human AP endonuclease 1. DOI:10.1016/j.dnarep.2004.06.009. PMID:15380100.
  4. Lowry DF et al. (2003), J Mol Biol, 329, 311-322. Investigation of the Role of the Histidine–Aspartate Pair in the Human Exonuclease III-like Abasic Endonuclease, Ape1. DOI:10.1016/s0022-2836(03)00382-6. PMID:12758078.
  5. Rothwell DG et al. (2000), Nucleic Acids Res, 28, 2207-2213. Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding. DOI:10.1093/nar/28.11.2207. PMID:10871340.
  6. Erzberger JP et al. (1999), J Mol Biol, 290, 447-457. The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: new insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis. DOI:10.1006/jmbi.1999.2888. PMID:10390343.

Step 1. Asp210 acts as a general base activating the water molecule for nucleophilic attack on the scissile phosphate. Asn212 positions the water for nucleophilic attack in a hydrogen bond network with Asp210. The pentaphosphate intermediate formed is stabilized by His309, Asp283, the Mg ions and Tyr171.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His309(278)A electrostatic stabiliser
Asp283(252)A electrostatic stabiliser
Asp70(39)A metal ligand
Glu96(65)A metal ligand
Tyr171(140)A metal ligand
Asp210(179)A metal ligand
Asp308(277)A metal ligand
His309(278)A metal ligand
Tyr171(140)A electrostatic stabiliser
Asp210(179)A increase nucleophilicity
Asp210(179)A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used

Step 2. 7,8-dihydro-8-oxoguanine is then cleaved from the phosphate, this cleavage is catalyzed through electrostatic stabilization.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp70(39)A metal ligand
Glu96(65)A metal ligand
Tyr171(140)A metal ligand
Asp210(179)A metal ligand
Asp308(277)A metal ligand
His309(278)A metal ligand
Tyr171(140)A electrostatic stabiliser
Asp283(252)A electrostatic stabiliser
His309(278)A electrostatic stabiliser

Chemical Components

ingold: intramolecular elimination, overall product formed
Download: Image, Marvin File

Step 1. Asp210 acts as a general base activating the water molecule for nucleophilic attack on the scissile phosphate. Asn212 positions the water for nucleophilic attack in a hydrogen bond network with Asp210. The pentaphosphate intermediate formed is stabilized by His309, Asp283, the Mg ions and Tyr171.

Step 2. 7,8-dihydro-8-oxoguanine is then cleaved from the phosphate, this cleavage is catalyzed through electrostatic stabilization.

Products.

Contributors

Fiona J. E. Morgan, James Willey