PDBsum entry: 1jn3

Transferase

PDB id: 1jn3

Contents

Protein chain

242 a.a. *

Waters ×185

* Residue conservation analysis

PDB id:

1jn3

Name:

Transferase

Title:

Fidelity properties and structure of m282l mutator mutant of polymerase: subtle structural changes influence the mechani nucleotide discrimination

Structure:

DNA polymerase beta. Chain: a. Fragment: catalytic domain, residues 85 - 335. Synonym: betanucleotidyltransferase. Beta polymerase. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.35Å R-factor: 0.215 R-free: 0.256

Authors:

D.A.Conn,J.B.Sweasy,J.Jaeger

Key ref:

A.M.Shah et al. (2001). A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability. Biochemistry, 40, 11372-11381. PubMed id: 11560485 DOI: 10.1021/bi010755y

Date:

22-Jul-01 Release date: 03-Aug-01

Protein chain

P06766  (DPOLB_RAT) -  DNA polymerase beta

Seq: 335 a.a.

Struc: 242 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: Deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1)

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

 Gene Ontology (GO) functional annotation 

• Biological process: DNA repair (1 term)
• Biochemical function: DNA binding (3 terms)

reference

DOI no: 10.1021/bi010755y

Biochemistry 40:11372-11381 (2001)

PubMed id: 11560485

A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability.

A.M.Shah, D.A.Conn, S.X.Li, A.Capaldi, J.Jäger, J.B.Sweasy.

ABSTRACT

DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity.