Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals
Localization of the active site of HIV-1 reverse transcriptase-associated RNase H domain on a DNA template using site-specific generated hydroxyl radicals
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DOI：
10.1074/jbc.273.17.10139
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发表时间：
1998-04-24
影响因子：
4.8
通讯作者：
Heumann, H
中科院分区：
文献类型：
--
作者：
Götte, M;Maier, G;Heumann, H
逆转录酶 (RT) 相关核糖核酸酶 H (RNase H) 可以切割 DNA/RNA 杂交体的 RNA 模板以及双链 (ds) RNA。该报告表明，当 HIV-RT 的 RNase H 活性位点中的 Mg2+ 被 Fe2+ 取代时，人类免疫缺陷病毒 (HIV)-RT 也可以切割 dsDNA 的模板链。切割机制以及切割位置根据使用的是 RNA 还是 DNA 而有所不同。 DNA 在引物 3' 端上游 17 个碱基位置被切割，RNA 在上游 18 个碱基位置被切割，竞争实验表明 Fe2+ 取代了 RT 相关 RNase H 的催化活性 Mg2+，结合的 Fe2+ 是局部生成的 OH 自由基的来源，可切割 DNA 中最接近的碱基。切割片段的电泳迁移率研究表明，DNA 通过氧化机制切割，而 RNA 通过酶促机制切割，这与 Mg2+ 依赖性切割没有区别。 Fe2+ 依赖性切割可用于追踪 dsDNA 以及 dsRNA 和 DNA/RNA 杂交体上 RT 相关 RNase H 的活性位点。 DNA 和 RNA 模板上切割位置观察到的 1 个碱基差异可归因于结合核酸的构象差异。我们认为，与 dsDNA 相比，dsRNA 和 DNA/RNA 杂交体的螺距较低，允许在引物 3' 端和 RNase H 活性位点处 Fe2+ 依赖性切割位置之间的区域容纳额外的碱基对。
Reverse transcriptase (RT)-associated ribonuclease H (RNase H) can cleave both the RNA template of DNA/RNA hybrids as well as double-stranded (ds) RNA. This report shows that human immunodeficiency virus (HIV)-RT can also cleave the template strand of dsDNA when Mg2+ is replaced by Fe2+ in the RNase H active site of HIV-RT. The cleavage mechanisms as well as the positions of the cut vary depending on whether RNA or DNA is used. While DNA is cleaved 17 base positions upstream of the primer 3'-end, RNA is cleaved 18 base positions upstream, Competition experiments show that Fe2+ replaces the catalytically active Mg2+ of RT-associated RNase H, The bound Fe2+ is the source of locally generated OH-radicals that cleave the most proximate base in the DNA. Electrophoretic mobility studies of the cleaved fragments suggest that DNA is cleaved by an oxidative mechanism, while RNA is cleaved by an enzymatic mechanism which is indistinguishable from the Mg2+-dependent cleavage. The Fe2+-dependent cuts can be used to trace the active site of RT-associated RNase H on dsDNA as well as on dsRNA and DNA/RNA hybrids. The observed 1 base difference in the cleavage positions on DNA and RNA templates can be attributed to conformational differences of the bound nucleic acids. We suggest that the lower pitch of dsRNA and DNA/RNA hybrids compared with dsDNA permits accommodation of an additional base pair in the region between the primer 3'-end and the Fe2+-dependent cleavage position at the RNase H active site.