Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.
Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.
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DOI：
10.1016/j.jmb.2009.11.072
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发表时间：
2010-02-05
影响因子：
5.6
通讯作者：
Xie XS
中科院分区：
文献类型：
--
作者：
Kim S;Schroeder CM;Xie XS
人类免疫缺陷病毒-1逆转录酶(HIV-1 RT)是一种多功能聚合酶，负责HIV基因组的逆转录，包括在RNA和DNA模板上的DNA复制。在体内逆转录过程中，HIV-1RT通过RNA和单链DNA模板上的各种二级结构进行复制，而不需要解开蛋白质的核酸，如解旋酶。为了通过二级结构了解聚合的机制，我们利用多重单分子DNA流动拉伸实验研究了HIV-1RT在长单链DNA模板上的DNA聚合活性。我们观察到，HIV-1RT在DNA单链区(18.7nt/S)进行快速引物延伸DNA合成，并将其活性切换到DNA发夹位置(2.3nT/S)的慢链置换合成。此外，我们还发现链置换合成的速度依赖于发夹茎中的GC含量和模板张拉力。这表明链置换合成是通过一种既不完全主动也不完全被动的机制进行的，即DNA发夹的打开是由dNTP水解过程中释放的自由能和碱基对的热磨损共同驱动的。我们的实验观察为HIV-1 RT在长单链DNA模板上进行DNA复制的互换模式提供了新的见解。
Human Immunodeficiency Virus-1 reverse transcriptase (HIV-1 RT) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long single-stranded DNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive, i.e. the opening of the DNA hairpin is driven by a combination of free energy released during dNTP hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long single-stranded DNA templates.
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影响因子：
2.9
作者：
LADUCA, RJ;FAY, PJ;BAMBARA, RA
通讯作者：
BAMBARA, RA
DOI：
10.1016/s0167-4781(99)00011-1
发表时间：
1999-03-19
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION
影响因子：
--
作者：
Klasens, BIF;Huthoff, HT;Berkhout, B
通讯作者：
Berkhout, B
影响因子：
4.8
作者：
Kelleher, CD;Champoux, JJ
通讯作者：
Champoux, JJ
影响因子：
2.9
作者：
Ji, XD;Klarmann, GJ;Preston, BD
通讯作者：
Preston, BD
影响因子：
4.8
作者：
Harris, D;Kaushik, N;Pandey, VN
通讯作者：
Pandey, VN