EUKARYOTIC DNA-POLYMERASE AMINO-ACID-SEQUENCE REQUIRED FOR 3'-] 5' EXONUCLEASE ACTIVITY
EUKARYOTIC DNA-POLYMERASE AMINO-ACID-SEQUENCE REQUIRED FOR 3'-] 5' EXONUCLEASE ACTIVITY
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DOI：
10.1073/pnas.88.21.9473
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发表时间：
1991-11-01
影响因子：
11.1
通讯作者：
SUGINO, A
中科院分区：
文献类型：
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作者：
MORRISON, A;BELL, JB;SUGINO, A
我们在酿酒酵母DNA聚合酶II中鉴定出一个氨基近端序列基序，即苯丙氨酸 - 天冬氨酸 - 异亮氨酸 - 谷氨酸 - 苏氨酸，它几乎与大肠杆菌DNA聚合酶I的3'→5'核酸外切酶活性位点的一部分序列相同。通过氨基酸序列比对，在具有3'→5'校对核酸外切酶活性的相关、对蚜栖菌素敏感的DNA聚合酶中也鉴定出了类似的基序。用丙氨酸替代该基序中的天冬氨酸和谷氨酸残基，在保留聚合酶活性的同时，使部分纯化的DNA聚合酶II的核酸外切酶活性至少降低了100倍。表达核酸外切酶缺陷型DNA聚合酶II的酵母菌株的自发突变率平均增加了约22倍，这与推测的其在体内的校对作用相符。在这个基序以及先前描述的另外两个保守基序的多氨基酸序列比对中，大肠杆菌DNA聚合酶I的3'→5'核酸外切酶活性位点的五个残基在已知具有3'→5'校对核酸外切酶活性的对蚜栖菌素敏感的DNA聚合酶中似乎是不变的。然而，在人、酵母或果蝇的α - DNA聚合酶的催化亚基中，这些残基似乎都无法识别。
We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3' --> 5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3' --> 5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3' --> 5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' --> 5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha-DNA polymerases.