Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides
Critical amino acids in human DNA polymerases η and κ involved in erroneous incorporation of oxidized nucleotides
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DOI：
10.1093/nar/gkp1095
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发表时间：
2010-01-01
影响因子：
14.9
通讯作者：
Nohmi, Takehiko
中科院分区：
文献类型：
--
作者：
Katafuchi, Atsushi;Sassa, Akira;Nohmi, Takehiko
当氧化的DNA前体掺入基因组中时，它们可以引起诱变和致癌。一些人Y家族DNA聚合酶（Pos）可以有效地将8-oxo-dGTP（dGTP的氧化形式）掺入到模板dA的相对位置。这种不适当的G：A配对可能导致A到C的颠换。为了深入了解错误核苷酸掺入的机制，我们改变了人类Pol eta和Pol kappa蛋白中的氨基酸，这些氨基酸可能调节它们将8-oxo-dGTP掺入DNA的特异性。我们发现Poleta中的Arg61对于错误的核苷酸掺入至关重要。当Arg61被赖氨酸（R61K）取代时，与dC配对相比，dA与8-氧代-dGTP配对的比率从660：1（野生型Pol eta）降低至7：1（R61K）。类似地，Pol kappa中的Tyr112对于错误的核苷酸掺入至关重要。当Tyr112被丙氨酸（Y112A）取代时，配对比率从11：1（野生型Pol κ）降低到几乎1：1（Y112A）。有趣的是，在Pol eta中相应位置的取代，即Phe18取代为丙氨酸，并没有改变特异性。这些结果表明，在不同位置的氨基酸在Pol eta和Pol kappa的活性位点可能会增强8-oxo-dGTP，有利于顺式构象，从而指导其错误地掺入DNA。
Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Pol eta and Pol kappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Pol eta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Pol eta) to 7 : 1 (R61K). Similarly, Tyr112 in Pol kappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Pol kappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Pol eta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Pol eta and Pol kappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.