ACCURACY OF DNA REPLICATION IN VITRO

我们感兴趣的是确定人体细胞 控制自发和诱发突变率。 我们先前已经 显示了人类细胞中主要的复制DNA聚合酶，当 纯化为相对简单的DNA聚合酶， 复杂性，在DNA合成反应中不够准确 在体外，以解释体内低自发突变率。 然而，众所周知，复制涉及 许多蛋白质的协同作用。 因此我们 检测保守性双向DNA的保真度 通过人HeLa细胞复制复合物进行复制。 我们最初 结果，在能够检测 各种错误，表明这个复制系统 至少比纯化的复制DNA精确20倍 单独的聚合酶。 这一意见得到了以下方面的证实： 几个控制实验，必要的复杂性质 该系统 为了更准确地评估 已经构建了诱变载体并用于 监测碱基替换和移码保真度 复制复合物在高度敏感的回复试验。 这些试验的初步结果表明，对于负一 重复模板序列的移码错误，DNA合成 复制复合体的精确度至少是 纯化的DNA聚合酶α。 然而，碱基替换 复制复合物的保真度虽然很高，但仍然是 不足以解释观察到的极低突变率 在完整的细胞中。 这表明，额外的机制， 维持精确的DNA合成的关键因素存在于细胞内。 我们目前正在解剖复制系统， 组成部分和测试的影响，个别亚单位 特定类型的突变事件使用高度敏感的 回复突变测定。 我们打算把重点放在有助于 保真度，包括碱基选择性，核酸外切 校对和复制后纠错。

We are interested in determing the mechanisms by which human cells control spontaneous and induced mutation rates. We have previously shown tht the major replicative DNA polymerase in human cells, when purified as a relatively simple DNA polymerase of limited complexity, is not accurate enough during DNA synthesis reactions in vitro to account for low spontaneous mutations rates in vivo. However, it is well established the replication involves the concerted action of a number of proteins. We are therefore examining the fidelity of semiconservative bidirectional DNA replication by a human HeLa cell replication complex. Our initial results, obtained in a forward mutation assay capable of detecting a variety of errors, indicating that this replication system was at least 20-fold more accurate than the purified replicative DNA polymerase alone. This observation has been substantiated by several control experiments, necessitated by the complicated nature of the system. In order to more accurately assess the fidelity of replication, mutagenesis vectors have been constructed and used to monitor the base substitution and frameshift fidelity of the replication complex in highly sensitive reversion assays. Initial results with these assays demonstrate that for minus-one frameshift errors at reiterated template sequences, DNA synthesis by the replication complex is at least 500-fold more accurate than purified DNA polymerase alpha. However, the base substitution fidelity of the replication complex, while high, is still insufficient to explain the extremely low mutation rates observed in intact cells. This suggests that additional mechanisms for maintaining accurate DNA synthesis are present within the cell. We are currently dissecting the replication system into its component parts and testing the effects of individual subunits on specific types of mutation events using the highly sensitive reversion assays. We intend to focus on mechanisms that contribute to fidelity, including base selectivity, exonucleolytic proofreading and post-replication error correction.