Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase
Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase
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DOI：
10.1074/jbc.m313759200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Choi, JY;Guengerich, FP
鸟嘌呤（G）的N-2原子易受各种致癌物的修饰。分析在该位置具有增加的体积的寡核苷酸的保真度和用进行性DNA聚合酶人类免疫缺陷病毒1型、逆转录酶（RT）和噬菌体T7核酸外切酶（T7（-））的催化效率。RT和T7（-）有效地绕过N-2-甲基（Me）G并容易延伸引物，但被N-2-乙基（Et）G、N-2-异丁基G、N-2-苄基G和N-2-甲基（9-蒽基）G强烈阻断。通过RT和T7（-）进行的单核苷酸掺入的稳态动力学显示，与N-2-MeG相反，dCTP掺入的k（cat）/K-m降低103，与N-2-EtG相反，降低幅度更大。Me组的错误掺入频率增加了10（2）~ 10（3）倍，Et组的错误掺入频率增加了10（3）倍。dATP优先结合在大体积N-2-烷基G分子的对面。N-2-MeG减弱RT和T7（-）的前稳态动力学爆发，N-2-EtG消除爆发。在N-2-EtG中观察到硫代-dCTP（α S）的大元素效应（6倍和72倍降低），但在N-2-MeG中则小得多，表明N-2-Et基团可能影响化学步骤（磷酸二酯键形成）的速率。RT和T7（-）的DNA底物的K-d（dCTP）和K-d（DNA）和k（off）速率的相似值表明基态结合和解离速率不受体积的显著影响。我们的结论是，即使是在鸟嘌呤N-2原子的Me基团可以引起一个深刻的干扰效果的保真度和效率; Et或更大的基团导致优先错误掺入和强大的阻断复制聚合酶，可能在化学步骤之前，展示了批量DNA损伤的作用。
The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease (T7(-)). RT and T7(-) effectively bypassed N-2-methyl(Me) G and readily extended primers but were strongly blocked by N-2-ethyl(Et)G, N-2-isobutylG, N-2-benzylG, and N-2-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 103 in k(cat)/K-m for dCTP incorporation opposite N-2-MeG and a further large decrease opposite N-2-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another similar to10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N-2-alkylG molecules. N-2-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N-2-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N-2-EtG (6-and 72-fold decreases) but were much less with N-2-MeG, indicating that the N-2-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K-d(dCTP) and K-d(DNA) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.