Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal.

Abstract:

DNA polymerases copy DNA templates with remarkably high fidelity, ...
DNA polymerases copy DNA templates with remarkably high fidelity, checking for correct base-pair formation both at nucleotide insertion and at subsequent DNA extension steps. Despite extensive biochemical, genetic and structural studies, the mechanism by which nucleotides are correctly incorporated is not known. Here we present high-resolution crystal structures of a thermostable bacterial (Bacillus stearothermophilus) DNA polymerase I large fragments with DNA primer templates bound productively at the polymerase active site. The active site retains catalytic activity, allowing direct observation of the products of several rounds of nucleotide incorporation. The polymerase also retains its ability to discriminate between correct and incorrectly paired nucleotides in the crystal. Comparison of the structures of successively translocated complexes allows the structural features for the sequence-independent molecular recognition of correctly formed base pairs to be deduced unambiguously. These include extensive interactions with the first four to five base pairs in the minor groove, location of the terminal base pair in a pocket of excellent steric complementarity favouring correct base-pair formation, and a conformational switch from B-form to underwound A-form DNA at the polymerase active site.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Structure and Structure/Function, Nucleotide Incorporation, Exonuclease Activity, Enzyme Substrate Interactions, Source / Purification

One line summary:

Crystal structure (1.8A) of the large fragment bound to primer templates (9bp duplex with 5base template overhang) and same after inclusion of the ddTTP (matching the first nucleotide in the overhang) to reveal the formation of the 10th pair of the duplex.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Polymerase Catalytic Residue Amino Acids Asp653,Asp830,Glu831
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. 3-5' Exonuclease (proofreading) No
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Cloned or native Cloned in E. coli
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. 5-3' Exonuclease Yes
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Full length or truncated Truncated
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Amino Acids Contacting Template Tyr714
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Processivity 111bp
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 1.5E+05 units/mg Technique: Polymerase Assay (calf thymus DNA)
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 4.9E+05 units/mg Technique: Polymerase Assay (M13 DNA)
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 13uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 3.4nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. kcat 191.2 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Bst LF Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Describe truncation contains aa 297-876
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. 3-5' Exonuclease (proofreading) No
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Full length or truncated Full length
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Processivity 10bp
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 2.2E+04 units/mg Technique: Polymerase Assay (calf thymus DNA)
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 4.9E+04 units/mg Technique: Polymerase Assay (M13 DNA)
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 3.5nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 24uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Taq pol I Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. kcat 46.6 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Polymerase Catalytic Residue Amino Acids Asp705, Asp882, and Glu883
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. 3-5' Exonuclease (proofreading) Yes
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Residues Involved in Catalysis of 3-5' Exo Asp355, Glu357, Asp424, and Asp501
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Processivity 70bp
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 5500 units/mg Technique: Polymerase Assay (M13 DNA)
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Specific Activity 9700 units/mg Technique: Polymerase Assay (calf thymus DNA)
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 2.3uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. KM 1.8nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Klenow fragment Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. kcat 5.2 /second Reaction: Nucleotide incorporation; Substrate: dNTPs

Entry validated by:

Structures:

3BDP 2BDP 4BDP

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