Aliotta J M, Pelletier J J, Ware J L, Moran L S, Benner J S, Kong H
New England Biolabs, Inc., Beverly Massachusetts 01915, USA.
Genet Anal. 1996 Mar;12(5-6):185-95.
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.
制备了来自嗜热脂肪芽孢杆菌N3468的一种热稳定DNA聚合酶——Bst DNA聚合酶I，纯度接近均一。在SDS聚丙烯酰胺凝胶上分析时，Bst DNA聚合酶I制剂的主要条带大小约为97 kDa。对编码Bst聚合酶I的Bst polA基因进行了克隆和测序。比较序列分析表明，在Bst DNA聚合酶I中不存在大肠杆菌DNA聚合酶I中发现的所有三个保守的3'→5'外切核酸酶基序。这使人怀疑该酶中是否存在3'→5'外切核酸酶功能。使用四种生化测定法来测量Bst DNA聚合酶I的外切核酸酶活性，测试全长Bst聚合酶I和缺乏N端5'→3'外切核酸酶结构域的Bst大片段。这些外切核酸酶测定表明，Bst DNA聚合酶I仅含有双链依赖性5'→3'外切核酸酶活性，但缺乏任何可检测到的3'→5'校对外切核酸酶活性。多种热稳定修复DNA聚合酶中缺乏3'→5'外切核酸酶功能可能反映出以校对活性为代价提高了热稳定性。
