PDBsum entry 5u9h

Transferase/DNA

PDB id: 5u9h

Contents

Protein chain

326 a.a.

DNA/RNA

Ligands

PPV

Metals

_MN ×4

_NA

Waters ×351

PDB id:

5u9h

Name:

Transferase/DNA

Title:

DNA polymerase beta product complex with inserted sp-isomer of dctp- alpha-s

Structure:

DNA (5'-d( Cp Cp Gp Ap Cp Gp Gp Cp Gp Cp Ap Tp Cp Ap Gp C)- 3'). Chain: t. Engineered: yes. DNA (5'-d( Gp Cp Tp Gp Ap Tp Gp Cp Gp Cp (C7r))-3'). Chain: p. Engineered: yes. DNA (5'-d(p Gp Tp Cp Gp G)-3'). Chain: d.

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Homo sapiens. Human. Organism_taxid: 9606. Gene: polb. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.85Å R-factor: 0.196 R-free: 0.233

Authors:

B.D.Freudenthal,S.H.Wilson,W.A.Beard

Key ref:

L.Perera et al. (2017). Revealing the role of the product metal in DNA polymerase β catalysis. Nucleic Acids Res, 45, 2736-2745. PubMed id: 28108654

Date:

16-Dec-16 Release date: 08-Feb-17

Protein chain

P06746  (DPOLB_HUMAN) -  DNA polymerase beta from Homo sapiens

Seq: 335 a.a.

Struc: 326 a.a.

DNA/RNA chains

C-C-G-A-C-G-G-C-G-C-A-T-C-A-G-C 16 bases

G-C-T-G-A-T-G-C-G-C-C7R 11 bases

G-T-C-G-G 5 bases

Enzyme reactions

• Enzyme class 1: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

DNA(n) + 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) (Bound ligand (Het Group name = PPV) corresponds exactly) + diphosphate

• Enzyme class 2: E.C.4.2.99.- - ?????
• Enzyme class 3: E.C.4.2.99.18 - DNA-(apurinic or apyrimidinic site) lyase.
• Reaction: 2'-deoxyribonucleotide-(2'-deoxyribose 5'-phosphate)- 2'-deoxyribonucleotide-DNA = a 3'-end 2'-deoxyribonucleotide-(2,3- dehydro-2,3-deoxyribose 5'-phosphate)-DNA + a 5'-end 5'-phospho- 2'-deoxyribonucleoside-DNA + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

Nucleic Acids Res 45:2736-2745 (2017)

PubMed id: 28108654

Revealing the role of the product metal in DNA polymerase β catalysis.

L.Perera, B.D.Freudenthal, W.A.Beard, L.G.Pedersen, S.H.Wilson.

ABSTRACT

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.