Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.
Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.
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脊椎动物 DNA 聚合酶 N 中残基的进化保守性赋予低保真度和旁路活性。
DOI：
10.1093/nar/gkq048
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发表时间：
2010
影响因子：
14.9
通讯作者：
Wood,RichardD
中科院分区：
文献类型：
--
作者：
Takata,Kei-ichi;Arana,MercedesE;Seki,Mineaki;Kunkel,ThomasA;Wood,RichardD
POLN 是脊椎动物基因组中编码的核 A 家族 DNA 聚合酶。 POLN 具有不寻常的保真度和 DNA 损伤旁路特性，包括强链置换活性、有利于 T 掺入模板 G 的低保真度以及通过 5S-胸腺嘧啶乙二醇 (5S-Tg) 的精确跨损伤合成。我们寻找聚合酶结构域的保守特征，以区别于原核 pol I 型 DNA 聚合酶。在指子域中基序 4 的保守“O 螺旋”中鉴定出特别令人感兴趣的赖氨酸残基（人 POLN 中的 679）。相应的残基是控制原核 pol I 保真度最重要的残基之一，并且是这些酶中的非极性丙氨酸或苏氨酸。动力学测量表明，K679A 或 K679T POLN 突变体 DNA 聚合酶对未损坏的模板具有完全活性，但很难掺入相对模板 G 的 T，并且不能有效绕过 5S-Tg。我们还发现同一基序中保守的Tyr残基不仅影响对双脱氧核苷酸的敏感性，而且极大地影响酶活性、保真度和旁路。蛋白质序列比对显示 POLN 在 DNA 聚合酶结构域中具有三个特定插入。结果表明，残基在进化过程中被严格保留，赋予 POLN 独特的旁路和保真度特性。
POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved ‘O-helix’ of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.