Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase γ
Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase γ
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DOI：
10.1074/jbc.m101114200
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发表时间：
2001-06-29
影响因子：
4.8
通讯作者：
Copeland, WC
中科院分区：
文献类型：
--
作者：
Lim, SE;Copeland, WC
线粒体毒性可导致用于控制人类免疫缺陷病毒1型感染的抗病毒核苷酸类似物治疗。我们通过比较六种抗病毒核苷酸类似物的插入和核酸外切酶去除，评估了这些类似物抑制人线粒体DNA聚合酶（pol γ）的DNA合成的能力。插入2 '，3' -双脱氧-TTP（ddTTP）、3 ' -叠氮基-TTP（AZT-TP）、2 '，3 '-双脱氧-TTP（dd-TTP）、2'，3 '-双脱氢-TTP（D4 T-TP）、（-）-2 '，3 ' -双脱氧-3' -硫代胞苷（3 TC-TP）和碳环2 '的表观稳态K-m和k（cat）值，3 ' -二脱氢-ddGTP（CBV-TP）表明所有六种类似物的掺入，尽管具有不同的效率。双脱氧核苷酸和D4 T-TP在体外与天然脱氧核苷酸一样有效地被聚合物γ利用，而AZT-TP、3 TC-TP和CBV-TP仅是DNA链延伸的中度抑制剂。双脱氧核苷酸，D4 T，AZT，和CBV从DNA的无效切除预测持久性在体内成功掺入后。相比之下，3 '末端3 TC残基的去除效率为天然3 '末端的50%。最后，我观察到已知在细胞中发生的AZT-单磷酸浓度对核酸外切酶活性的抑制。因此，尽管它们的最大抑制作用是通过掺入和链终止，但这些类似物在DNA中的持久性和对核酸外切校正的抑制也可能导致线粒体毒性。
Mitochondrial toxicity can result hom antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K-m and k(cat) values for insertion of 2 ' ,3 ' -dideoxy-TTP (ddTTP), 3 ' -azido-TTP (AZT-TP), 2 ' ,3 ' -dideoxy-TTP (dd-TTP), 2 ' ,3 ' -didehydro-TTP (D4T-TP), (-)-2 ' ,3 ' -dideoxy-3 ' -thiacytidine (3TC-TP), and carbocyclic 2 ' ,3 ' -didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3 ' termini. Finally, me observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.