PDBsum entry 1lv5

Transferase/DNA

PDB id: 1lv5

Contents

Protein chains

580 a.a. *

DNA/RNA

Ligands

SO4 ×2

DCP ×2

Metals

_MN ×2

_MG

Waters ×186

* Residue conservation analysis

PDB id:

1lv5

Name:

Transferase/DNA

Title:

Crystal structure of the closed conformation of bacillus DNA polymerase i fragment bound to DNA and dctp

Structure:

5'-d( Gp Gp Ap Tp Cp Ap Gp Cp Gp A)-3'. Chain: c, e. Engineered: yes. Other_details: primer strand. 5'-d( Ap Cp Gp Tp Cp Gp Cp Tp Gp Ap Tp Cp Cp G)-3'. Chain: d, f. Engineered: yes. Other_details: template strand. DNA polymerase i.

Source:

Synthetic: yes. Geobacillus stearothermophilus. Organism_taxid: 1422. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: this protein was isolated from an as yet unnamed novel strain of bacillus stearothermophilus (see ref 2).

Biol. unit:

Hexamer (from PQS)

Resolution:

1.95Å R-factor: 0.218 R-free: 0.261

Authors:

S.J.Johnson,J.S.Taylor,L.S.Beese

Key ref:

S.J.Johnson et al. (2003). Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations. Proc Natl Acad Sci U S A, 100, 3895-3900. PubMed id: 12649320 DOI: 10.1073/pnas.0630532100

Date:

24-May-02 Release date: 25-Mar-03

Protein chains

P52026  (DPO1_GEOSE) -  DNA polymerase I from Geobacillus stearothermophilus

Seq: 876 a.a.

Struc: 580 a.a.*

* PDB and UniProt seqs differ at 67 residue positions (black crosses)

DNA/RNA chains

G-G-A-T-C-A-G-C-G-A 10 bases

A-C-G-T-C-G-C-T-G-A-T-C-C-G 14 bases

G-G-A-T-C-A-G-C-G-A 10 bases

A-C-G-T-C-G-C-T-G-A-T-C-C-G 14 bases

Enzyme reactions

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

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

Added reference

DOI no: 10.1073/pnas.0630532100

Proc Natl Acad Sci U S A 100:3895-3900 (2003)

PubMed id: 12649320

Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations.

S.J.Johnson, J.S.Taylor, L.S.Beese.

ABSTRACT

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

Selected figure(s)

Figure 2.
Fig. 2. Active site superposition of open and closed BF structures. (A) The 11-bp open binary complex (yellow) and the closed ternary complex (blue) are shown in stereo view. The largest conformational differences occur in the fingers domain, including the O helix, O1 helix, and preinsertion site. The acceptor template base (n) occupies the preinsertion site in the open conformation and the insertion site in the closed conformation. (B) A close-up view of the preinsertion site. The locations of the conserved Tyr-714 are indicated.

Figure 3.
Fig. 3. Conformational interlocks during DNA synthesis. A schematic overview of the polymerase active site (A) and atomic coordinates (B) derived from the open and closed BF structures represent a complete round of DNA synthesis. The conformational changes described here are presented in animated form in Movie 1, which is published as supporting information on the PNAS web site. The reaction cycle starts with the acceptor template base (n, red) bound at the template preinsertion site (between the O and O1 helices; green shading); Tyr-714 blocks access to the insertion site (blue shading) and stacks with the n-1 base pair at the postinsertion site (gray shading). Formation of the closed conformation involves rearrangement of the O and O1 helices, which simultaneously blocks the template preinsertion site and unblocks the insertion site. These rearrangements move the acceptor template base (n) to the insertion site, where it pairs with an incoming dNTP (green). Nucleotide incorporation occurs on formation of a cognate base pair and proper assembly of the catalytic site (orange shading). The cycle is completed with translocation of the DNA by one base pair position. The polymerase resets to the open conformation in preparation for the next round of DNA synthesis.

Figures were selected by an automated process.