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

Abstract:

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:

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

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 Kiefer JR1998 Polymerase Catalytic Residue Amino Acids Asp653,Asp830,Glu831
Bst LF Kiefer JR1998 3-5' Exonuclease (proofreading) No
Bst LF Kiefer JR1998 Cloned or native Cloned in E. coli
Bst LF Kiefer JR1998 5-3' Exonuclease Yes
Bst LF Kiefer JR1998 Full length or truncated Truncated
Bst LF Kiefer JR1998 Amino Acids Contacting Template Tyr714
Bst LF Kiefer JR1998 Processivity 111bp
Bst LF Kiefer JR1998 Specific Activity 1.5E+05 units/mg Technique: Polymerase Assay (calf thymus DNA)
Bst LF Kiefer JR1998 Specific Activity 4.9E+05 units/mg Technique: Polymerase Assay (M13 DNA)
Bst LF Kiefer JR1998 KM 13uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Bst LF Kiefer JR1998 KM 3.4nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Bst LF Kiefer JR1998 kcat 191.2 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Bst LF Kiefer JR1998 Describe truncation contains aa 297-876
Taq pol I Kiefer JR1998 3-5' Exonuclease (proofreading) No
Taq pol I Kiefer JR1998 Full length or truncated Full length
Taq pol I Kiefer JR1998 Processivity 10bp
Taq pol I Kiefer JR1998 Specific Activity 2.2E+04 units/mg Technique: Polymerase Assay (calf thymus DNA)
Taq pol I Kiefer JR1998 Specific Activity 4.9E+04 units/mg Technique: Polymerase Assay (M13 DNA)
Taq pol I Kiefer JR1998 KM 3.5nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Taq pol I Kiefer JR1998 KM 24uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Taq pol I Kiefer JR1998 kcat 46.6 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Kiefer JR1998 Polymerase Catalytic Residue Amino Acids Asp705, Asp882, and Glu883
Klenow fragment Kiefer JR1998 3-5' Exonuclease (proofreading) Yes
Klenow fragment Kiefer JR1998 Residues Involved in Catalysis of 3-5' Exo Asp355, Glu357, Asp424, and Asp501
Klenow fragment Kiefer JR1998 Processivity 70bp
Klenow fragment Kiefer JR1998 Specific Activity 5500 units/mg Technique: Polymerase Assay (M13 DNA)
Klenow fragment Kiefer JR1998 Specific Activity 9700 units/mg Technique: Polymerase Assay (calf thymus DNA)
Klenow fragment Kiefer JR1998 KM 2.3uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Klenow fragment Kiefer JR1998 KM 1.8nM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Klenow fragment Kiefer JR1998 kcat 5.2 /second Reaction: Nucleotide incorporation; Substrate: dNTPs

Entry validated by:

Structures:

3BDP 2BDP 4BDP
Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.