Ishino Y, Takahashi-Fujii A, Uemori T, Imamura M, Kato I, Doi H
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga, Japan.
Protein Eng. 1995 Nov;8(11):1171-5. doi: 10.1093/protein/8.11.1171.
We studied the 5' --> 3' exonuclease activity of Bacillus caldotenax DNA polymerase by site-directed mutagenesis. Among seven mutants constructed, two mutant DNA polymerases with an amino acid substitution of Gly184 --> Asp or Gly192 --> Asp were confirmed to be deficient in this exonuclease. The two positions corresponded to those of the Escherichia coli DNA polymerase I mutants defective in 5' --> 3' exonuclease, polA480ex and polA214. These results provide experimental support for the proposed amino acid sequence essential for the 5' --> 3' exonuclease activity associated with eubacterial polymerase I-like DNA polymerases (family A), including E.coli and Thermus aquaticus.
我们通过定点诱变研究了嗜热栖热放线菌DNA聚合酶的5'→3'核酸外切酶活性。在所构建的7个突变体中，证实有两个氨基酸发生Gly184→Asp或Gly192→Asp取代的突变DNA聚合酶缺乏这种核酸外切酶活性。这两个位置与大肠杆菌DNA聚合酶I中5'→3'核酸外切酶缺陷型突变体polA480ex和polA214的相应位置一致。这些结果为与真细菌聚合酶I样DNA聚合酶（A族）相关的5'→3'核酸外切酶活性所必需的氨基酸序列提供了实验支持，这些聚合酶包括大肠杆菌和嗜热水生栖热菌的DNA聚合酶。
