Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity.

Abstract:

A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus ...
A thermostable DNA polymerase, the Bst DNA polymerase I, from Bacillus stearothermophilus N3468 was prepared to near-homogeneity. The dominant species of the Bst DNA polymerase I preparation sized about 97 kDa when analyzed on SDS polyacrylamide gels. The Bst polA gene that codes for Bst polymerase I was cloned and sequenced. Comparative sequence analysis showed that all three conserved 3'-->5' exonuclease motifs found in E. coli DNA polymerase I were missing in Bst DNA polymerase I. This cast doubt on the existence of a 3'-->5' exonuclease function in that enzyme. Four biochemical assays were used to measure exonuclease activities of Bst DNA polymerase I, testing both full-length Bst polymerase I and the Bst large fragment which lacks the N-terminal 5'-->3' exonuclease domain. These exonuclease assays demonstrated that Bst DNA polymerase I only contained a double-strand dependent 5'-->3' exonuclease activity but lacked any detectable 3'-->5' proofreading exonuclease activity. The lack of 3'-->5' exonuclease function in a variety of thermostable repair DNA polymerases may reflect enhancement of thermostability at the expense of proofreading activity.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Exonuclease Activity, Source / Purification

One line summary:

This paper clarifies controversial descriptions of exonuclease activity of Bst polymerase

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Molecular Weight 99.01kD Technique: Sequence
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. 3-5' Exonuclease (proofreading) No
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Cloned or native Native organism
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. 5-3' Exonuclease Yes
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Full length or truncated Full length
Bst Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Specific Activity 1.5E+05 units/mg
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Molecular Weight 67kD Technique: SDS-PAGE (Cleaved from MBP tag)
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. 3-5' Exonuclease (proofreading) No
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Cloned or native Cloned in E. coli
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. 5-3' Exonuclease No
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Tagged Yes
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Tag Name MBP
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Full length or truncated Truncated
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Specific Activity 1E+05 units/mg
Bst LF Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity. Describe truncation Lacks 5'->3' exonuclease domain det by subtilisin digestion - deletion up to nt 867

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