Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors
Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors
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DOI：
10.1093/emboj/16.4.856
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发表时间：
1997-02-17
期刊：
影响因子：
11.4
通讯作者：
Telesnitsky, A
中科院分区：
文献类型：
--
作者：
Kulpa, D;Topping, R;Telesnitsky, A
在逆转录病毒DNA合成过程中，逆转录酶必须执行两个专门的模板切换。在这里，我们使用Moloney鼠白血病病毒为基础的载体来检测细胞内反转录过程中其中一个开关的位置。与原始的逆转录模型一致，但与之前的实验数据相反，我们观察到第一链转移几乎总是准确地发生在基因组RNA的5‘端。这一发现使我们能够使用第一链转移来研究逆转录酶在病毒DNA合成过程中在指定位置切换模板时可能和/或确实会犯的错误类别。我们发现，错误发生在第一链转移类似的位置，比报道的模板内部位置的平均逆转录酶错误率高出1000倍。然后，我们分析了专门载体的复制产物，这些载体被设计来测试开关相关错误的可能来源。我们的结果表明，至少有一些错误是通过非模板核苷酸加成，然后在链转移点错配延伸而产生的。我们讨论了我们的发现的意义，因为它们与模板切换相关的错误可能对逆转录病毒突变率的可能贡献有关。
Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5' end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer similar to 1000-fold more frequently than reported average reverse transcriptase error rates for template-internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch-associated errors. Our results suggest that at least some errors arose via nontemplated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch-associated errors may make to retroviral mutation rates.