Misalignment-mediated DNA polymerase β mutations:: Comparison of microsatellite and frame-shift error rates using a forward mutation assay
Misalignment-mediated DNA polymerase β mutations:: Comparison of microsatellite and frame-shift error rates using a forward mutation assay
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DOI：
10.1021/bi025918c
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发表时间：
2002-08-20
期刊：
影响因子：
2.9
通讯作者：
Hile, SE
中科院分区：
文献类型：
--
作者：
Eckert, KA;Mowery, A;Hile, SE
微卫星 DNA 中发生的突变与神经系统疾病和癌症有关。为了阐明微卫星突变的分子基础，我们确定了代表人类基因组中发现的微卫星序列的体外聚合酶错误频率：[GT/CA](10)、[TC/AG](11) 和 [TTCC/AAGG](9)。 DNA 模板包含符合读码框插入单纯疱疹病毒胸苷激酶 (HSV-tk) 基因 5' 区域的微卫星。相对于来自相同 DNA 合成反应的编码序列中的移码错误频率，对微卫星序列中的聚合酶 β (polbeta) 错误频率进行定量。二核苷酸序列内的polbeta错误频率为(2-9) x 10(-3)，比ssDNA模板频率高14-72倍。四核苷酸序列内的 polbeta 错误频率为 (4-6) x 10(-3)，比背景增加了 4-13 倍。观察到 [TC/AG](11) 和 [TTCC/AAGG](9) 等位基因的链偏倚，其中当嘌呤链作为模板时产生更多错误。每个微卫星内的突变包括非规范碱基替换事件和单核苷酸缺失以及预期的单位长度变化。在每个位点的聚合酶错误频率与重复单元的数量和等位基因的总长度之间观察到指数关系。我们的观察结果与微卫星诱变的链滑移模型一致，并证明 DNA 序列和/或结构差异导致突变链偏差。据我们所知，这是首次使用模板微卫星序列对 DNA 聚合酶错误进行体外直接定量。
Mutations arising in microsatellite DNA are associated with neurological diseases and cancer. To elucidate the molecular basis of microsatellite mutation, we have determined the in vitro polymerase error frequencies at microsatellite sequences representative of those found in the human genome: [GT/CA](10), [TC/AG](11), and [TTCC/AAGG](9). DNA templates contained the microsatellites inserted in-frame into the 5' region of the herpes simplex virus thymidine kinase (HSV-tk) gene. Polymerase beta (polbeta) error frequencies were quantitated in microsatellite sequences, relative to frame-shift error frequencies in coding sequences, from the same DNA synthesis reaction. The polbeta error frequencies within the dinucleotide sequences were (2-9) x 10(-3), 14-72-fold higher than the ssDNA template frequencies. The polbeta error frequencies within the tetranucleotide sequences were (4-6) x 10(-3), a 4-13-fold increase over background. Strand biases were observed for the [TC/AG](11) and [TTCC/AAGG](9) alleles, in which more errors were produced when the purine strand served as a template. Mutations within each microsatellite included noncanonical base substitution events and single nucleotide deletions as well as the expected unit length changes. An exponential relationship was observed between the polymerase error frequency per site and both the number of repetitive units and total length of the allele. Our observations are consistent with the strand slippage model of microsatellite mutagenesis and demonstrate that DNA sequence and/or structural differences result in mutational strand biases. To our knowledge, this is the first direct quantitation of DNA polymerase errors in vitro using template microsatellite sequences.