PDBsum entry 4m9h

Transferase/DNA

PDB id: 4m9h

Contents

Protein chain

314 a.a.

DNA/RNA

Ligands

TTP

Metals

_NA ×2

_CL

Waters ×223

PDB id:

4m9h

Name:

Transferase/DNA

Title:

DNA polymerase beta e295k soaked with dttp

Structure:

DNA polymerase beta. Chain: a. Engineered: yes. Mutation: yes. DNA template strand. Chain: t. Engineered: yes. DNA primer strand. Chain: p.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: polb. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: DNA template strand, 16mer. Other_details: DNA primer strand, 10mer.

Resolution:

2.39Å R-factor: 0.182 R-free: 0.228

Authors:

B.E.Eckenroth,S.Doublie

Key ref:

B.E.Eckenroth et al. (2013). The E295K cancer variant of human polymerase β favors the mismatch conformational pathway during nucleotide selection. J Biol Chem, 288, 34850-34860. PubMed id: 24133209 DOI: 10.1074/jbc.M113.510891

Date:

14-Aug-13 Release date: 16-Oct-13

Protein chain

P06746  (DPOLB_HUMAN) -  DNA polymerase beta from Homo sapiens

Seq: 335 a.a.

Struc: 314 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

C-C-G-A-C-A-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

DNA(n) + 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) (Bound ligand (Het Group name = TTP) matches with 69.23% similarity) + 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: 10.1074/jbc.M113.510891

J Biol Chem 288:34850-34860 (2013)

PubMed id: 24133209

The E295K cancer variant of human polymerase β favors the mismatch conformational pathway during nucleotide selection.

B.E.Eckenroth, J.B.Towle-Weicksel, J.B.Sweasy, S.Doublié.

ABSTRACT

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.