期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 395, 期 5, 页码 995-1006出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.11.072
关键词
HIV-1 reverse transcriptase; single molecule; DNA flow-stretching assay; DNA hairpin; strand displacement synthesis
资金
- Jane Coffin Childs Memorial Fund
- National Institutes of Health
HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase) is a Multifunctional polymerase responsible for reverse transcription of the HI V 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 (ssDNA) 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 ssDNA 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: that is, the opening of the DNA hairpin is driven by a combination of free energy released during dNTP (deoxyribonucleotide triphosphate) 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 ssDNA templates. (C) Published by Elsevier Ltd.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据