PDBsum entry 4ubc

Transferase, lyase/DNA

PDB id: 4ubc

Contents

Protein chain

326 a.a.

DNA/RNA

Ligands

8DG

Metals

_CA ×3

Waters ×302

PDB id:

4ubc

Name:

Transferase, lyase/DNA

Title:

DNA polymerase beta substrate complex with a templating cytosine and incoming 8-oxodgtp, 0 s

Structure:

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

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:

2.00Å R-factor: 0.190 R-free: 0.260

Authors:

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

Key ref:

B.D.Freudenthal et al. (2015). Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature, 517, 635-639. PubMed id: 25409153 DOI: 10.1038/nature13886

Date:

12-Aug-14 Release date: 12-Nov-14

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

G-C-T-G-A-T-G-C-G-C 10 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
• 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

DOI no: 10.1038/nature13886

Nature 517:635-639 (2015)

PubMed id: 25409153

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide.

B.D.Freudenthal, W.A.Beard, L.Perera, D.D.Shock, T.Kim, T.Schlick, S.H.Wilson.

ABSTRACT

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.