Department of Biochemistry, University of Western Ontario, London, ON, Canada N6A 5C1.
Proc Natl Acad Sci U S A. 2011 Feb 22;108(8):3210-5. doi: 10.1073/pnas.1013909108. Epub 2011 Feb 7.
The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.
8-氧鸟嘌呤（8-oxo-G）损伤是基因组中存在的最丰富和最具诱变的氧化 DNA 损伤。由于其双重编码性质，8-oxo-G 导致大多数 DNA 聚合酶错误掺入腺嘌呤。人类 Y 家族 DNA 聚合酶iota（polι）优先在 8-oxo-G 对面掺入正确的胞嘧啶核苷酸。这种独特的特异性可能有助于 polι 在细胞抵御氧化应激中的生物学作用。然而，这种优先掺入胞嘧啶的结构基础目前尚不清楚。在这里，我们展示了 polι 与含有 8-oxo-G 损伤的 DNA 复合物的四个晶体结构，与正确的 dCTP 或错误的 dATP、dGTP 和 dTTP 核苷酸配对。一个异常狭窄的 polι 活性位点将嘌呤碱基限制在一个顺式构象中，通过抑制顺/反构象平衡来阻止 8-oxo-G 的双重编码特性。更重要的是，在 polι 中，顺式 8-oxo-G 碱基没有诱变作用，因为其 Hoogsteen 边缘不能在狭窄的活性位点中与 dATP 形成稳定的碱基对。相反，由于其嘧啶碱基较小且与 8-oxo-G 的 Hoogsteen 边缘增强氢键，顺式 8-oxo-G 模板碱基与正确的 dCTP 形成最稳定的复制碱基对。结合定点突变，我们表明，指状结构域中的 Gln59 特异性地与损伤碱基的额外 O(8)原子相互作用，这影响核苷酸选择、酶效率和损伤部位的复制停滞。我们的工作提供了人类 DNA 聚合酶高保真 8-oxo-G 复制的结构机制。
