Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled.
Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled.
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DOI：
10.1016/s0021-9258(20)89464-2
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发表时间：
1991-04
期刊：
影响因子：
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通讯作者：
J. DeStefano;R. Buiser;L. Mallaber;Thomas W. Myers;R. Bambara;P. Fay
中科院分区：
文献类型：
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作者：
J. DeStefano;R. Buiser;L. Mallaber;Thomas W. Myers;R. Bambara;P. Fay
使用在特定位置引发的272个核苷酸长的质粒衍生的RNA转录本，检查RNA依赖性DNA聚合酶和逆转录酶（RT）的RNase H活性之间的功能相互作用。研究了禽成髓细胞瘤病毒（AMV）RT、人免疫缺陷病毒RT和莫洛尼鼠白血病病毒RT的特性。所有三种酶与引物-模板形成稳定的复合物，半衰期范围为约16至41秒。每种酶合成全长引物延伸产物，并在DNA合成期间切割RNA模板至少一次。然后在一轮进行性DNA合成后有效地隔离RT的挑战RNA存在下测定聚合。该测定允许测量在与引物-模板的一次接触期间由RT催化的内切核酸裂解的数目。结果表明，无论是否存在脱氧核苷三磷酸以允许合成，三种RT中的每一种都在从引物-模板解离之前切割转录物。在合成过程中，RNA降解的程度在RT中不同，AMV-RT主要产生大片段的RNA-DNA杂合物，几乎没有小RNA切割产物。人类免疫缺陷病毒和莫洛尼鼠白血病病毒-RT比AMV-RT产生更多的小降解产物，但仍然留下了许多潜在的可降解的混合物未消化。结果表明，RNase H的功能是远远低于聚合功能的活性在进行性DNA合成，活动并不严格耦合。
The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.