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


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.



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

One line summary:

This paper clarifies controversial descriptions of exonuclease activity of Bst polymerase


new topics/pols set partial results complete validated


Polymerase Reference Property Result Context
Bst Aliotta JM1996 Molecular Weight 99.01kD Technique: Sequence
Bst Aliotta JM1996 3-5' Exonuclease (proofreading) No
Bst Aliotta JM1996 Cloned or native Native organism
Bst Aliotta JM1996 5-3' Exonuclease Yes
Bst Aliotta JM1996 Full length or truncated Full length
Bst Aliotta JM1996 Specific Activity 1.5E+05 units/mg
Bst LF Aliotta JM1996 Molecular Weight 67kD Technique: SDS-PAGE (Cleaved from MBP tag)
Bst LF Aliotta JM1996 3-5' Exonuclease (proofreading) No
Bst LF Aliotta JM1996 Cloned or native Cloned in E. coli
Bst LF Aliotta JM1996 5-3' Exonuclease No
Bst LF Aliotta JM1996 Tagged Yes
Bst LF Aliotta JM1996 Tag Name MBP
Bst LF Aliotta JM1996 Full length or truncated Truncated
Bst LF Aliotta JM1996 Specific Activity 1E+05 units/mg
Bst LF Aliotta JM1996 Describe truncation Lacks 5'->3' exonuclease domain det by subtilisin digestion - deletion up to nt 867

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