Strand transfer mediated by human immunodeficiency virus reverse transcriptase in vitro is promoted by pausing and results in misincorporation.
Strand transfer mediated by human immunodeficiency virus reverse transcriptase in vitro is promoted by pausing and results in misincorporation.
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体外由人类免疫缺陷病毒逆转录酶介导的链转移通过暂停而被促进并导致错误掺入。
DOI：
10.1074/jbc.270.1.325
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Bambara,RA
中科院分区：
文献类型：
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作者：
Wu,W;Blumberg,BM;Fay,PJ;Bambara,RA
人类免疫缺陷病毒(HIV-1)能够通过将生长的DNA链从一个基因组的内部区域转移到另一个基因组进行重组。链转移反应是在HIV-1逆转录酶(RT)催化下，在天然HIV-1 nef基因的供体和受体RNA模板之间进行的。供体和受体模板共享一个可能发生链转移的几乎同源的区域，不同之处仅在于受体与供体相比有36个核苷酸插入和6个大间距的碱基替换。我们对经过转移的细长引物进行了测序。从供体序列到受体序列的变化揭示了转移的位置。结果表明，RT在合成过程中暂停的位置与链转移的增强呈正相关。在序列变化最小的情况下消除停顿位点，可减少紧邻区域的链转移频率。对在常用位点转移产生的DNA产物的序列分析表明，在转移点左右的DNA中引入了突变。值得注意的是，大约30%的产品含有突变。观察到碱基替换、短增加和短缺失。在没有转移的情况下，在供体模板上延伸的DNA产物中没有出现突变。这些突变的一致性表明，它们是由滑移和非模板导向的核苷酸加成共同引起的。这些结果表明，所检测到的突变与链转移过程有关。
Human immunodeficiency virus (HIV-1) is able to recombine by transfer of the growing DNA strand from internal regions of one genome to another. The strand transfer reaction, catalyzed by HIV-1 reverse transcriptase (RT), was conductedin vitrobetween donor and acceptor RNA templates that were derived from natural HIV-1nefgenes. The donor and acceptor templates shared a nearly homologous region where strand transfer could occur, differing only in that the acceptor had a 36-nucleotide insertion and 6 widely spaced base substitutions compared with the donor. We sequenced elongated primers that underwent transfer. The position of transfer was revealed by the change of sequence from that of the donor to that of the acceptor. Results showed a positive correlation between positions where the RT paused during synthesis and enhancement of strand transfer. Elimination of a pause site, with a minimal change in sequence, decreased the frequency of strand transfer in the immediate area.Analysis of the sequence of DNA products resulting from transfer at a frequently used site showed that mutations had been introduced into the DNA at about the point of transfer. Remarkably, approximately 30% of the products contained mutations. Base substitutions, short additions and deletions were observed. Mutations did not appear in DNA products extended on the donor template without transfer. The identity of the mutations suggests that they were caused by a combination of slippage and non-template-directed nucleotide addition. These results indicated that the detected mutations were related to the process of strand transfer.