Variants of mouse DNA polymerase. reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a] pyrene dG adduct
Variants of mouse DNA polymerase. reveal a mechanism of efficient and accurate translesion synthesis past a benzo[a] pyrene dG adduct
复制标题
小鼠 DNA 聚合酶的变体。
DOI：
10.1073/pnas.1324168111
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发表时间：
2014-02-04
影响因子：
11.1
通讯作者：
Guo, Caixia
中科院分区：
文献类型：
--
作者：
Liu, Yang;Yang, Yeran;Guo, Caixia
DNA聚合酶kappa(Pol Kappa)是目前已知的唯一一种能高效、准确地绕过10S(+)-反式反式苯并[a]芘二醇环氧化物(BPDE)-N-2-脱氧鸟嘌呤加合物的Y-家族DNA聚合酶。Pol kappa的独特功能，催化核心和小指域之间的大结构间隙以及N末端90个残基的添加被称为N-CLAP，这可能是其特殊的跨损伤能力的原因。我们设计并构建了两个小鼠Pol kappa变异体，它们在模板碱基两侧[Pol kappa Gap Mutant(PGM)1]或一侧(PGM2)的间隙变小。这些Pol kappa变异体在正常DNA合成中几乎与WT一样有效，尽管准确度有所降低。然而，PGM1被10S(+)-反式抗BPDE-N-2-DG损伤强烈阻断。稳态动力学测量显示，PGM1对损伤的dCTP掺入效率显著降低，而PGM2的掺入效率略有降低。稳定补充了PGM1 GFP-Pol kappa的Pol.缺陷细胞对BPDE处理保持高度敏感，而补充WT或PGM2 GFP-Pol kappa则恢复了BPDE抗性。此外，小鼠Pol kappa(mPol(kappa 52-516))N-CLASP的前51个残基的缺失导致聚合活性降低，突变体PGM2(52-516)而不是PGM1(52-516)可以部分补偿N-末端的缺失，恢复对正常DNA的催化活性。然而，WT和PGM2 mPol(kappa 52-516)都不保留BPDE旁路活动。我们得出结论，结构间隙在物理上容纳了大体积的芳香族加合物，而N-环对于POL的结构完整性和灵活性是必不可少的。在跨病变合成过程中。
DNA polymerase kappa (Pol kappa) is the only known Y-family DNA polymerase that bypasses the 10S (+)-trans-anti-benzo[a] pyrene diol epoxide (BPDE)-N-2-deoxyguanine adducts efficiently and accurately. The unique features of Pol kappa, a large structure gap between the catalytic core and little finger domain and a 90-residue addition at the N terminus known as the N-clasp, may give rise to its special translesion capability. We designed and constructed two mouse Pol kappa variants, which have reduced gap size on both sides [Pol kappa Gap Mutant (PGM) 1] or one side flanking the template base (PGM2). These Pol kappa variants are nearly as efficient as WT in normal DNA synthesis, albeit with reduced accuracy. However, PGM1 is strongly blocked by the 10S (+)-trans-anti-BPDE-N-2-dG lesion. Steady-state kinetic measurements reveal a significant reduction in efficiency of dCTP incorporation opposite the lesion by PGM1 and a moderate reduction by PGM2. Consistently, Pol.-deficient cells stably complemented with PGM1 GFP-Pol kappa remained hypersensitive to BPDE treatment, and complementation with WT or PGM2 GFP-Pol kappa restored BPDE resistance. Furthermore, deletion of the first 51 residues of the N-clasp in mouse Pol kappa (mPol(kappa 52-516)) leads to reduced polymerization activity, and the mutant PGM2(52-516) but not PGM1(52-516) can partially compensate the N-terminal deletion and restore the catalytic activity on normal DNA. However, neither WT nor PGM2 mPol(kappa 52-516) retains BPDE bypass activity. We conclude that the structural gap physically accommodates the bulky aromatic adduct and the N-clasp is essential for the structural integrity and flexibility of Pol. during translesion synthesis.