Bebenek K, Roberts J D, Kunkel T A
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1992 Feb 25;267(6):3589-96.
The use of unequal concentrations of the four deoxynucleoside triphosphates (dNTPs) in DNA polymerization reactions alters base substitution error rates in a predictable way. Less is known about the effects of substrate imbalances on base addition and deletion error rates. Thus, we examined pool bias effects on frameshift fidelity during DNA synthesis catalyzed by replicative DNA polymerases. Imbalanced pools altered the frameshift fidelity of the human immunodeficiency virus type-1 reverse transcriptase. Both mutagenic and antimutagenic effects were observed for minus-one, plus-one, and minus-two nucleotide errors, in a highly sequence-specific manner. Most of this specificity can be rationalized by either of two models. One involves frameshifts initiated by pool bias-induced nucleotide misinsertion, and the other involves pool bias-initiated template-primer slippage. Several examples of complex mutations were also recovered more than once in small mutant collections. These contained closely spaced single-base substitution and minus-one base frameshift changes. The two changes occurred at a frequency much higher than predicted if they were generated independently. This suggests that when the polymerase makes one mistake, the probability that it will make a second mistake within the next few incorporations increases significantly. Perturbation of dNTP pools also affected the frameshift fidelity of the replicative yeast DNA polymerase alpha. In reactions containing a low concentration of one dNTP, the error rate increased for one-nucleotide deletions at homopolymeric template nucleotides complementary to the dNTP whose concentration was low. We extended this approach to determine the frameshift fidelity of simian virus 40 origin-dependent semiconservative replication of double-stranded DNA in extracts of human cells. In reactions performed with an equal concentration of all four dNTPs, replication was highly accurate for minus-one-nucleotide errors. However, when the concentration of one dNTP was decreased, the replication error rate increased at complementary, homopolymeric template positions. This response provides an approach for describing frameshift accuracy during replication of the leading and lagging strands.
在DNA聚合反应中使用浓度不等的四种脱氧核苷三磷酸（dNTP），会以一种可预测的方式改变碱基替换错误率。关于底物失衡对碱基添加和缺失错误率的影响，人们了解得较少。因此，我们研究了在复制性DNA聚合酶催化的DNA合成过程中，底物库偏差对移码保真度的影响。不平衡的底物库改变了人类免疫缺陷病毒1型逆转录酶的移码保真度。对于减一、加一和减二核苷酸错误，均观察到了诱变和抗诱变效应，且具有高度的序列特异性。这种特异性的大部分可以通过两种模型中的任何一种来解释。一种涉及由底物库偏差诱导的核苷酸错插入引发的移码，另一种涉及底物库偏差引发的模板 - 引物滑动。在小型突变体库中，还多次发现了几个复杂突变的例子。这些包含紧密间隔的单碱基替换和减一碱基移码变化。这两种变化发生的频率远高于它们独立产生时的预测频率。这表明当聚合酶犯一个错误时，在接下来的几次掺入中犯第二个错误的概率会显著增加。dNTP底物库的扰动也影响了复制性酵母DNA聚合酶α的移码保真度。在含有低浓度一种dNTP的反应中，与浓度低的dNTP互补的同聚模板核苷酸处的单核苷酸缺失错误率增加。我们扩展了这种方法，以确定在人细胞提取物中双链DNA的猿猴病毒40起源依赖性半保留复制的移码保真度。在使用所有四种dNTP等浓度进行的反应中，对于减一核苷酸错误，复制非常准确。然而，当一种dNTP的浓度降低时，在互补的同聚模板位置，复制错误率会增加。这种反应为描述前导链和后随链复制过程中的移码准确性提供了一种方法。
