Structural determinants in human DNA polymerase γ account for mitochondrial toxicity from nucleoside analogs
Structural determinants in human DNA polymerase γ account for mitochondrial toxicity from nucleoside analogs
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DOI：
10.1016/s0022-2836(03)00405-4
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发表时间：
2003-05-23
影响因子：
5.6
通讯作者：
Copeland, WC
中科院分区：
文献类型：
--
作者：
Lim, SE;Ponamarev, MV;Copeland, WC
虽然抗病毒核苷类似物疗法成功地延缓了艾滋病毒感染向艾滋病的进展，但这些药物通过诱导线粒体毒性而产生了不受欢迎的副作用。我们和其他人已经证明了线粒体聚合酶，DNA聚合酶伽马(PolGamma)，通过将这些链终止的抗病毒核苷酸类似物结合到DNA中，参与线粒体毒性。在这里，我们探索了三个高度保守的氨基酸残基在人类Pol伽马活性部位的作用，它们调节了核苷酸类似物的选择作为掺入的底物。DNA聚合酶的序列比对、晶体结构和诱变研究导致我们将聚合酶基序B中的Tyr951和Tyr955改变为Phe和Ala，将聚合酶A中的Glu895改变为Ala。测试了突变聚合酶结合天然核苷酸和目前被批准用于抗病毒治疗的五种抗病毒核苷类似物：AZT、ddC、d4T、3TC和卡韦的能力。POL伽马衍生物与正常核苷酸和抗病毒核苷酸的稳态动力学分析表明，Tyr951在很大程度上是POL伽马结合双脱氧核苷酸和d4T-MP的能力的原因。Tyr951突变为Phe，使该酶对双脱氧核苷酸和d4T-TP产生抗药性，而不影响聚合酶的活性。在核苷酸正常的情况下，Glu895和Tyr955改变为Ala对聚合酶总活性的影响最大，导致K-m(DNTP)显著增加，k(CAT)显著下降。PolGamma中Tyr955的突变导致人类退行性疾病进行性外眼肌麻痹，我们表明这一残基部分解释了PolGamma结合d4T-MP和卡韦的能力。Glu895对ALA的改变略微增加了对双脱氧核苷酸和d4T-TP的识别能力。讨论了波尔伽马选择某些核苷酸类似物的机制。爱思唯尔科学有限公司出版。
Although antiviral nucleoside analog therapy successfully delays progression of HIV infection to AIDS, these drugs cause unwelcome side-effects by inducing mitochondrial toxicity. We and others have demonstrated that the mitochondrial polymerase, DNA polymerase gamma (pol gamma), participates in mitochondrial toxicity by incorporating these chain-terminating antiviral nucleotide analogs into DNA. Here, we explore the role of three highly conserved amino acid residues in the active site of human pol gamma that modulate selection of nucleotide analogs as substrates for incorporation. Sequence alignments, crystal structures and mutagenesis studies of family A DNA polymerases led us to change Tyr951 and Tyr955 in polymerase motif B to Phe and Ala, and Glu895 in polymerase motif A was changed to Ala. The mutant polymerases were tested for their ability to incorporate natural nucleotides and the five antiviral nucleoside analogs currently approved for antiviral therapy: AZT, ddC, D4T, 3TC and carbovir. Steady-state kinetic analysis of the pol gamma derivatives with the normal and antiviral nucleotides demonstrated that Tyr951 is largely responsible for the ability of pol gamma to incorporate dideoxynucleotides and D4T-MP. Mutation of Tyr951 to Phe renders the enzyme resistant to dideoxynucleotides and D4T-TP without compromising the activity of the polymerase. Alteration of Glu895 and Tyr955 to Ala had the largest effect on overall polymerase activity with normal nucleotides, producing dramatic increases in K-m(dNTP) and large decreases in k(cat). Mutation of Tyr955 in pol gamma causes the degenerative disease progressive external ophthalmoplegia in humans, and we show that this residue partially accounts for the ability of pol gamma to incorporate D4T-MP and carbovir. Alteration of Glu895 to Ala slightly increased discrimination against dideoxynucleotides and D4T-TP. The mechanisms by which pol gamma selects certain nucleotide analogs are discussed. Published by Elsevier Science Ltd.