DNA nucleotidylexotransferase

 

Terminal deoxynucleotidyl transferase (TdT) catalyses the condensation of deoxyribonucleotide triphosphates onto the 3' hydroxyl ends of DNA strands in a template-independent manner. It can also catalyse the addition of ribonucleotides and a range of unnatural nucleotides onto DNA strands. TdT has only been found in mammals, where it is highly conserved. It has a role in the generation of combinatorial diversity in lymphocytes, where it functions to add nucleotides (N regions) to the V(D)J recombination junctions of immunoglobulin and T-cell receptor genes. Together with DNA polymerase beta and lambda, TdT belongs to a family of polymerases called pol X, a subclass of an ancient nucleotidyl transferase (NT) superfamily. The active site of this family is structurally similar to that of the pol I and pol alpha families even though the topology of the catalytic domain is different; in all cases the active site is made up of three carboxylate side chains which bind two divalent cations. Tdt is unique in this respect due to its ability to be catalytically active with a range of divalent cations bound, Co2+, Mn2+, Zn2+and Mg2+. The ion bound determines substrate specificity and the enzyme's kinetics.

 

Reference Protein and Structure

Sequence
P09838 UniProt (2.7.7.31, 3.1.11.-) IPR027292 (Sequence Homologues) (PDB Homologues)
Biological species
Mus musculus (house mouse) Uniprot
PDB
1jms - Crystal Structure of the Catalytic Core of Murine Terminal Deoxynucleotidyl Transferase (2.36 Å) PDBe PDBsum 1jms
Catalytic CATH Domains
3.30.460.10 CATHdb (see all for 1jms)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:2.7.7.31)

2'-deoxyribonucleoside 5'-triphosphate(4-)
CHEBI:61560ChEBI
+
adenosine 5'-monophosphate
CHEBI:16027ChEBI
→
diphosphate(3-)
CHEBI:33019ChEBI
+
DNA polyanion
CHEBI:83828ChEBI
Alternative enzyme names: TdT, Addase, Deoxynucleotidyl terminal transferase, Deoxyribonucleic acid nucleotidyltransferase, Deoxyribonucleic nucleotidyltransferase, Terminal addition enzyme, Terminal deoxynucleotide transferase, Terminal deoxyribonucleotidyltransferase, Terminal deoxynucleotidyltransferase, Terminal transferase,

Enzyme Mechanism

Introduction

By analogy with DNA polymerase beta, TdT uses an Mg2+ ion to promote attack by the 3' hydroxyl group of the primer on the alpha phosphorous of the incoming NTP which is coupled to the stereochemical inversion. Coordination of the 3' hydroxyl group to the Mg2+ ion lowers the pKa of this group to allow deprotonation by Asp 434 during attack on the NTP. The Mg2+ ion also provides a positive charge to stabilise the pentacoordinate transition state of the alpha phosphorous.

Catalytic Residues Roles

UniProt PDB* (1jms)
Asp343, Asp345 Asp343(214)A, Asp345(216)A Interacts with divalent metal ions. metal ligand
Asp434 Asp434(305)A Accepts proton from attacking 3' OH group of primer. metal ligand, proton acceptor, proton donor
*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

bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used, inferred reaction step, native state of enzyme regenerated

References

  1. Delarue M et al. (2002), EMBO J, 21, 427-439. Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase. DOI:10.1093/emboj/21.3.427. PMID:11823435.
  2. Beard WA et al. (2014), Biochemistry, 53, 2768-2780. Structure and mechanism of DNA polymerase β. DOI:10.1021/bi500139h. PMID:24717170.
  3. Bebenek K et al. (2014), Biochemistry, 53, 2781-2792. Structure-function studies of DNA polymerase λ. DOI:10.1021/bi4017236. PMID:24716527.
  4. Motea EA et al. (2010), Biochim Biophys Acta, 1804, 1151-1166. Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase. DOI:10.1016/j.bbapap.2009.06.030. PMID:19596089.
  5. Cisneros GA et al. (2008), DNA Repair (Amst), 7, 1824-1834. Catalytic mechanism of human DNA polymerase lambda with Mg2+ and Mn2+ from ab initio quantum mechanical/molecular mechanical studies. DOI:10.1016/j.dnarep.2008.07.007. PMID:18692600.
  6. Boulé JB et al. (2001), J Biol Chem, 276, 31388-31393. Terminal Deoxynucleotidyl Transferase Indiscriminately Incorporates Ribonucleotides and Deoxyribonucleotides. DOI:10.1074/jbc.m105272200. PMID:11406636.
  7. Pelletier H et al. (1994), Science, 264, 1891-1903. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. DOI:10.2210/pdb2bpg/pdb. PMID:7516580.

Step 1. Mg2+A lowers the pKa around the 3'OH on RNA, facilitating protonation of Asp434. This inevitably increases the 3'OH nucleophilicity to attack the alpha phosphate on the incoming DTP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp343(214)A metal ligand
Asp345(216)A metal ligand
Asp434(305)A metal ligand
Asp434(305)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used

Step 2. Deprotonation of Asp434 by a water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp434(305)A proton donor

Chemical Components

proton transfer, inferred reaction step, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Mg2+A lowers the pKa around the 3'OH on RNA, facilitating protonation of Asp434. This inevitably increases the 3'OH nucleophilicity to attack the alpha phosphate on the incoming DTP.

Step 2. Deprotonation of Asp434 by a water molecule.

Products.

Contributors

Hannah Gilbert, Steven Smith, Gemma L. Holliday, Morwenna Hall