Thermostable Bst DNA polymerase I lacks a 3'-->5' proofreading exonuclease activity.
| Autoři: | Aliotta JM; New England Biolabs, Inc., Beverly Massachusetts 01915, USA., Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H |
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| Zdroj: | Genetic analysis : biomolecular engineering [Genet Anal] 1996 Mar; Vol. 12 (5-6), pp. 185-95. |
| Způsob vydávání: | Comparative Study; Journal Article |
| Jazyk: | English |
| Informace o časopise: | Publisher: Elsevier Country of Publication: Netherlands NLM ID: 9509403 Publication Model: Print Cited Medium: Print NLM ISO Abbreviation: Genet Anal Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Amsterdam ; New York : Elsevier, 1995-c1999. |
| Výrazy ze slovníku MeSH: | DNA Polymerase I/*metabolism, Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA Polymerase I/genetics ; DNA Polymerase I/isolation & purification ; DNA Primers/genetics ; DNA Repair ; DNA, Bacterial/genetics ; DNA, Bacterial/metabolism ; Enzyme Stability ; Escherichia coli/enzymology ; Escherichia coli/genetics ; Exodeoxyribonuclease V ; Exodeoxyribonucleases/genetics ; Exodeoxyribonucleases/isolation & purification ; Exodeoxyribonucleases/metabolism ; Genes, Bacterial ; Geobacillus stearothermophilus/enzymology ; Geobacillus stearothermophilus/genetics ; Molecular Sequence Data ; Molecular Weight ; Sequence Homology, Amino Acid ; Temperature |
| Abstrakt: | 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. |
| Molecular Sequence: | GENBANK U33536 |
| Substance Nomenclature: | 0 (DNA Primers) 0 (DNA, Bacterial) EC 2.7.7.7 (DNA Polymerase I) EC 3.1.- (Exodeoxyribonucleases) EC 3.1.11.5 (Exodeoxyribonuclease V) |
| Entry Date(s): | Date Created: 19960301 Date Completed: 19961021 Latest Revision: 20201209 |
| Update Code: | 20231215 |
| PMID: | 8740835 |
| Autor: | Aliotta JM; New England Biolabs, Inc., Beverly Massachusetts 01915, USA., Pelletier JJ, Ware JL, Moran LS, Benner JS, Kong H |
| Jazyk: | angličtina |
| Zdroj: | Genetic analysis : biomolecular engineering [Genet Anal] 1996 Mar; Vol. 12 (5-6), pp. 185-95. |
| Abstrakt: | 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. |
| Databáze: | MEDLINE |
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