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首页> 外文期刊>Molecular and Cellular Biology >Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.
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Fidelity of two retroviral reverse transcriptases during DNA-dependent DNA synthesis in vitro.

机译：在体外依赖DNA的DNA合成过程中两个逆转录病毒逆转录酶的保真度。

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We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.
机译：我们使用M13mp2 lacZ alpha基因作为突变靶点，在体外DNA合成过程中确定了禽成纤维细胞病病毒和莫洛尼鼠白血病病毒逆转录酶（RTs）的保真度。对于每聚合的30,000个核苷酸，两个RT都会犯一次错误。在能够检测多种类型错误的正向突变测定法中产生的突变体的DNA序列分析表明，禽成纤维细胞病病毒RT产生了多种不同的突变。多数（58％）是​​单碱基取代;所有这些都是由于dAMP或dGMP的错误掺入所致。负一移码也很常见，约占突变的30％。除单碱基事件外，八个突变体还包含2到59个碱基的序列变化。这些突变体的频率表明，至少在体外DNA合成过程中，RT还会通过除经典碱基错误编码和错位之外的机制引起错误。我们检查了RTs从错配引物末端以错配碱基与模板中下一个碱基互补的序列合成DNA的能力。与从错配的模板引物聚合的细胞DNA聚合酶不同，RT优选从模板链中包含匹配的末端碱基对和未配对碱基的重排模板引物聚合。对这种底物的不同寻常偏爱表明RT与模板引物之间的相互作用与细胞DNA聚合酶之间的相互作用不同。体外RT的总体错误率足以说明这些病毒的估计突变率。

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