Incorporation of uracil into minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription
Incorporation of uracil into minus strand DNA affects the specificity of plus strand synthesis initiation during lentiviral reverse transcription
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DOI：
10.1074/jbc.m207223200
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发表时间：
2003-03-07
影响因子：
4.8
通讯作者：
Preston, BD
中科院分区：
文献类型：
--
作者：
Klarmann, GJ;Chen, X;Preston, BD
许多逆转录病毒要么编码dUTP热磷酸酶（dUTPase），要么包装宿主源性尿嘧啶DNA糖基化酶，以限制尿嘧啶在DNA链中的积累，这表明尿嘧啶对病毒生命周期中的一个或多个步骤有害。在本研究中，在无细胞系统中研究了DNA尿尿对（-）链DNA合成、RNase H活性和（+）链DNA合成的影响。该系统利用纯化的人类免疫缺陷病毒1型（HIV-1）逆转录酶的活性，在单一反应混合物中将单链RNA转化为双链DNA。dUTP取代dTTP对（-）链的合成没有影响，但显著降低（+）链DNA的产率。新生（+）链5'端图谱显示，这是由于多嘌呤束起始减少而非多嘌呤束起始增加所致。当检测含有未激活DNA的预形成RNA-DNA双链时，异常起始与RNase H切割特异性的变化相关，这表明（+）链引物的适当“选择”受到影响。总的来说，这些数据表明逆转录病毒DNA中尿嘧啶的积累可能通过改变逆转录过程中（+）链DNA合成起始的特异性来破坏病毒的生命周期。
Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.