Polypurine tract primer generation and utilization by moloney murine leukemia virus reverse transcriptase
Polypurine tract primer generation and utilization by moloney murine leukemia virus reverse transcriptase
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DOI：
10.1074/jbc.274.49.34547
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发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Champoux, JJ
中科院分区：
文献类型：
--
作者：
Schultz, SJ;Zhang, MH;Champoux, JJ
在逆转录过程中，逆转录酶的RNaseH活性特异性地裂解病毒基因组中的多嘌呤途径(PPT)，产生用于启动正链DNA合成的引物，并非特异性地裂解病毒基因组，以促进正链DNA的合成。为了了解引物长度和序列如何影响PPT的产生和利用，我们使用了含有或不含有PPT的短杂交底物来评估逆转录酶的切割、延伸和结合。反转录酶同样能很好地延伸含有启动正链合成的3‘端RNA的底物，但引物长度影响对RNaseH裂解的敏感性。具有3‘端延伸到正链起始点以外的RNA底物延伸不良，但被特异性切割以产生正确的3’端以启动正链合成，含有PPT缺失的RNA的底物被非特异性切割且延伸效率低下。与破坏PPT引物或降解其他短RNA片段的切割相比，产生正链引物的特定切割和5‘端定向切割在动力学上更受青睐。PPT不抵抗分离的RNaseH结构域的切割，而分离的含有PPT序列的聚合酶结构域扩展的RNA引物与引物3‘端无关。这些结果为逆转录酶如何产生并选择性地利用PPT引物启动正链DNA合成提供了深入的见解。
During reverse transcription, the RNase H activity of reverse transcriptase specifically cleaves the viral genome within the polypurine tract (PPT) to create the primer used for the initiation of plus-strand DNA synthesis and nonspecifically cleaves the viral genome to facilitate synthesis of plus-strand DNA. To understand how primer length and sequence affect generation and utilization of the PPT, we employed short hybrid substrates containing or lacking the PPT to evaluate cleavage, extension, and binding by reverse transcriptase. Substrates containing RNAs with the correct 3' end for initiation of plus-strand synthesis were extended equally well by reverse transcriptase, but primer length affected susceptibility to RNase H cleavage. RNA substrates with 3' ends extending beyond the plus strand initiation site were extended poorly but were specifically cleaved to generate the correct 3' end for initiation of plus-strand synthesis, Substrates containing RNAs lacking the PPT were cleaved nonspecifically and extended inefficiently. Specific cleavages to generate the plus-strand primer and 5'-end-directed cleavages were kinetically favored over cleavages that destroyed the PPT primer or degraded other short RNA fragments. The PPT was not intrinsically resistant to cleavage by the isolated RNase H domain, and the isolated polymerase domain extended RNA primers containing the PPT sequence irrespective of the primer 3' end. These results provide insights into how reverse transcriptase generates and selectively utilizes the PPT primer for initiation of plus strand DNA synthesis.