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首页> 外文期刊>Mutation Research: International Journal on Mutagenesis, Chromosome Breakage and Related Subjects >Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro.
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Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro.

机译：生物DNA前体库不对称性对体外DNA复制准确性的影响。

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Deoxyguanosine triphosphate is underrepresented among the four common deoxyribonucleoside triphosphates (dNTPs), typically accounting for just 5-10% of the total dNTP pool. We have asked whether this pool asymmetry affects the fidelity of DNA replication, by use of an in vitro assay in which an M13 phagemid containing the Escherichia coli lacZalpha gene and an SV40 replication origin is replicated by extracts of human cells. By monitoring reversion of either a TGA or TAA codon within the lacZalpha gene, we found that replication in "biologically biased" dNTPs, representing our estimate of the concentrations in HeLa cell nuclei, is not significantly more accurate than when measured in reaction mixtures containing the four dNTPs at equimolar concentrations. However, sequence analysis of revertants revealed significantly different patterns of mispairing events leading to mutation. During replication at biased dNTP levels, mutations at the site 5' to C in the template strand for the TGA triplet were less frequent than seen in equimolar reaction mixtures, suggesting that extension from mismatches at this site is relatively slow, and proofreading efficiency high, when dGTP is the next nucleotide to be incorporated. Mismatches opposite template C, which might have been favored by the low physiological concentrations of dGTP, were not favored in our in vitro system, although one particular substitution at this site, TGA-->TTA, was strongly favored at low [dGTP]. An excess of one dNTP was found in our system to be more mutagenic than a corresponding deficiency. We also estimated dNTP concentrations in non-transformed human fibroblasts and found that in vitro replication at these levels caused significantly fewer mutations than we observed under equimolar conditions (100 microM each dNTP). This increased replication fidelity may result from increased proofreading efficiency at the lower dNTP levels; however, replication rates were decreased only slightly at these non-transformed fibroblast concentrations.
机译：在四种常见的脱氧核糖核苷三磷酸（dNTP）中，脱氧鸟苷三磷酸的含量不足，通常仅占总dNTP库的5-10％。我们已经问过这种池的不对称性是否通过使用体外测定方法来影响DNA复制的保真度，在该方法中，人细胞提取物复制了包含大肠杆菌lacZalpha基因和SV40复制起点的M13噬菌粒。通过监测lacZalpha基因中TGA或TAA密码子的回复，我们发现“生物学偏向” dNTPs中的复制（代表我们对HeLa细胞核浓度的估计）并不比在含有HLA细胞核的反应混合物中测量时准确得多。等摩尔浓度的四个dNTP。但是，对回复序列的序列分析显示导致突变的错配事件的模式显着不同。在dNTP偏向水平复制过程中，与等摩尔反应混合物相比，TGA三联体模板链中5'到C位点的突变频率要低，这表明该位点错配的延伸相对较慢，并且校对效率很高，当dGTP是要掺入的下一个核苷酸时。与模板C相对的错配可能由低生理浓度的dGTP引起，但在我们的体外系统中却不被接受，尽管在此[dGTP]低时强烈推荐该位点的一种特殊取代，TGA-> TTA。在我们的系统中发现过量的一种dNTP比相应的缺陷更具致突变性。我们还估计了未转化的人类成纤维细胞中的dNTP浓度，发现在这些水平下的体外复制所产生的突变比在等摩尔条件下观察到的突变少得多（每个dNTP为100 microM）。复制保真度的提高可能是由于在较低的dNTP级别提高了校对效率;但是，在这些未转化的成纤维细胞浓度下，复制率仅略有下降。

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