PDBsum entry 2isp

Transferase/DNA

PDB id: 2isp

Contents

Protein chain

326 a.a. *

DNA/RNA

Ligands

GGH

Metals

_MG

_NA ×3

_CL ×5

Waters ×302

* Residue conservation analysis

PDB id:

2isp

Name:

Transferase/DNA

Title:

Ternary complex of DNA polymerase beta with a dideoxy terminated primer and 2'-deoxyguanosine 5'-beta, gamma-methylene triphosphate

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 (Doc))-3'. Chain: p. Engineered: yes. 5'-d(p Gp Tp Cp Gp G)-3'. Chain: d. Engineered: yes.

Source:

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

Resolution:

2.20Å R-factor: 0.201 R-free: 0.261

Authors:

C.A.Sucato,T.G.Upton,B.A.Kashemirov,V.Martinek,Y.Xiang,W.A.Beard

Key ref:

C.A.Sucato et al. (2007). Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta. Biochemistry, 46, 461-471. PubMed id: 17209556 DOI: 10.1021/bi061517b

Date:

18-Oct-06 Release date: 30-Jan-07

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-DOC 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: 10.1021/bi061517b

Biochemistry 46:461-471 (2007)

PubMed id: 17209556

Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta.

C.A.Sucato, T.G.Upton, B.A.Kashemirov, V.K.Batra, V.Martínek, Y.Xiang, W.A.Beard, L.C.Pedersen, S.H.Wilson, C.E.McKenna, J.Florián, A.Warshel, M.F.Goodman.

ABSTRACT

DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.