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.
Aliotta
J M
JM
New England Biolabs, Inc., Beverly Massachusetts 01915, USA.
Pelletier
J J
JJ
Ware
J L
JL
Moran
L S
LS
Benner
J S
JS
Kong
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D017931:DNA Primers; D004269:DNA, Bacterial; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V