QUANTITATIVE-ANALYSIS OF RNA CLEAVAGE DURING RNA-DIRECTED DNA-SYNTHESIS BY HUMAN IMMUNODEFICIENCY AND AVIAN-MYELOBLASTOSIS VIRUS REVERSE TRANSCRIPTASES
QUANTITATIVE-ANALYSIS OF RNA CLEAVAGE DURING RNA-DIRECTED DNA-SYNTHESIS BY HUMAN IMMUNODEFICIENCY AND AVIAN-MYELOBLASTOSIS VIRUS REVERSE TRANSCRIPTASES
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DOI：
10.1093/nar/22.18.3793
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发表时间：
1994-09-11
影响因子：
14.9
通讯作者：
BAMBARA, RA
中科院分区：
文献类型：
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作者：
DESTEFANO, JJ;MALLABER, LM;BAMBARA, RA
我们已经确定了人类免疫缺陷病毒 (HIV) 或禽成髓细胞瘤病毒 (AMV) 逆转录酶 (RT) 在 DNA 合成过程中进行 RNA 切割的程度。选择允许在给定模板引物上的一次结合事件期间由 RT 进行切割和合成分析的条件。通过用大肠杆菌 RNase H 处理，确定 RT 合成后留下的核糖核酸酶 H (RNase H) 敏感模板 RNA 的最大数量。对预期太短而无法保持杂交（长度小于 13 个核苷酸）的 RNA 裂解产物进行了定量。结果表明，HIV-RT 和 AMV-RT 分别降解了这些短产物中约 80% 和不到 20% 的潜在可降解 RNA。较长的杂交 RNA 的存活并不是选择性丧失 RNase H 活性的 RT 群体合成的结果。通过评估引物延伸与 RT 在引物延伸过程中切割的 RNA 模板的比例，我们确定基本上每个带有聚合酶的 HIV-和 AMV-RT 分子也具有 RNase H 活性。结果表明，尽管 HIV-RT 和 AMV-RT 在合成过程中都会切割 RNA 模板，但 HIV-RT 添加的每个核苷酸的切割次数要多得多。他们还表明，一些杂交的 RNA 片段在 RT 通过后仍然保留，形成第一条 DNA 链。在体内，这些片段必须在随后的反应中被切割或移位，然后才能完成第二链 DNA 的合成。
We have determined the extent of RNA cleavage carried out during DNA synthesis by either human immunodeficiency virus (HIV) or avian myeloblastosis virus (AMV) reverse transcriptases (RTs). Conditions were chosen that allowed the analysis of the cleavage and synthesis performed by the RT during one binding event on a given template-primer. The maximum quantity of ribonuclease H (RNase H) sensitive template RNA left after synthesis by the RTs was determined by treatment with Escherichia coli RNase H. RNA cleavage products that were expected to be too short to remain hybridized, less than 13 nucleotides in length, were quantitated. Results showed that HIV-and AMV-RT degraded about 80% and less than 20%, respectively, of the potentially degradable RNA to these short products. Survival of longer, hybridized RNA was not a result of synthesis by a population of RTs that had selectively lost RNase H activity. Using an assay that evaluated the proportion of primers extended versus RNA templates cleaved during primer-extension by the RTs, we determined that essentially each molecule of HIV- and AMV-RT with polymerase also has RNase H activity. The results indicate that although both HIV- and AMV-RTs cleave the RNA template during synthesis, the number of cleavages per nucleotide addition with HIV-RT is much greater. They also suggest that some hybridized RNA segments remain right after the passage of the RT making the first DNA strand. In vivo, these segments would have to be cleaved or displaced in later reactions before second strand DNA synthesis could be completed.