Characterization of RNA strand displacement synthesis by moloney murine leukemia virus reverse transcriptase
Characterization of RNA strand displacement synthesis by moloney murine leukemia virus reverse transcriptase
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DOI：
10.1074/jbc.273.16.9976
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发表时间：
1998-04-17
影响因子：
4.8
通讯作者：
Champoux, JJ
中科院分区：
文献类型：
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作者：
Kelleher, CD;Champoux, JJ
逆转录酶（RT）的RNase H活性可能是在负链合成后切割RNA基因组以释放DNA用作正链合成过程中的模板所必需的。然而，由于RNA酶H的RNA降解似乎产生的RNA片段太大，不能自发地从负链上解离，我们已经研究了在正链合成过程中通过RT置换RNA有助于去除RNA片段的可能性。通过使用莫洛尼鼠白血病病毒（M-MuLV）RT的RNase H-突变体，我们证明了聚合酶可以用DNA置换杂交双链体中的RNA长区域，但这种活性比DNA置换慢约20倍，比非置换合成慢20倍。此外，我们发现，虽然某些杂交序列似乎几乎难治的RNA置换的启动，相同的序列可能不会显着阻碍合成时，由单链间隙之前。我们发现，野生型M-MuLV RT的RNA置换合成速率显著大于RNA酶H-RT，但仍低于非置换合成速率。M-MuLV核衣壳蛋白使RNA和DNA置换合成的速率增加约2倍，并且这种活性似乎需要锌指结构域。
The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H- mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 20-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H- RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.