DNA polymerase active site is highly mutable: Evolutionary consequences
DNA polymerase active site is highly mutable: Evolutionary consequences
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DOI：
10.1073/pnas.97.10.5095
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发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Loeb, LA
中科院分区：
文献类型：
--
作者：
Patel, PH;Loeb, LA
DNA聚合酶含有结构上可重叠且在序列上高度保守的活性位点。为了评估这种保存的意义并确定活性位点可以耐受的突变负荷，我们随机突变了栖热菌DNA聚合酶I的聚合酶催化位点（基序A）内的13个氨基酸。经过选择，通过使用遗传互补，我们获得了约8，000个活性突变DNA聚合酶的文库，其中350个进行了测序和分析。这是生理活性聚合酶突变体的最大集合。我们发现，所有残基的基序A，除了一个（天冬氨酸-610），是可变的，同时保留野生型活性。在进化上维持的位点处获得了各种各样的氨基酸取代，并且保守取代在稳定三级结构的区域占主导地位。几种突变体表现出独特的性质，包括DNA聚合酶活性高于野生型酶或掺入核糖核苷酸类似物的能力。依赖于这些突变的聚合酶生存的细菌适合重复复制。体内聚合酶活性位点的高突变性和进化改变的酶的能力可能是在需要增加诱变的环境中生存所必需的。相对于自然界中发现的核苷酸序列的恒定性，必须解决聚合酶活性位点的固有可取代性。
DNA polymerases contain active sites that are structurally super-imposable and highly conserved in sequence. To assess the significance of this preservation and to determine the mutational burden that active sites can tolerate, we randomly mutated a stretch of 13 amino acids within the polymerase catalytic site (motif A) of Thermus aquaticus DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8,000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all residues of motif A, except one (Asp-610), are mutable while preserving wild-type activity. A wide Variety of amino acid substitutions were obtained at sites that are evolutionarily maintained, and conservative substitutions predominate at regions that stabilize tertiary structures. Several mutants exhibit unique properties, including DNA polymerase activity higher than the wild-type enzyme or the ability to incorporate ribonucleotide analogs. Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. The high mutability of the polymerase active site in vivo and the ability to evolve altered enzymes may be required for survival in environments that demand increased mutagenesis. The inherent substitutability of the polymerase active site must be addressed relative to the constancy of nucleotide sequence found in nature.