ANALYSIS OF MOLECULAR MECHANISMS OF MUTAGENESIS WITH DEFINED COMPONENTS

复制和维护遗传信息的稳定性 需要DNA的精确合成。在动物细胞中，DNA合成是 由四种不同类别的DNA聚合酶执行， 德尔塔和伽马。我们的目标一直是描述 每种酶的DNA合成并分析误差 试图理解突变率是如何 控制住了。发现线粒体的高保真度 鸡的复制DNA聚合酶伽马是核外溶解的结果 校对，我们搜索并在哺乳动物中发现了类似的活动 来自两个来源的伽马聚合酶。这概括了对 与这类聚合酶相关的校对活动，以及 确定了四类高等真核DNA中的两类 聚合酶通过校对机制实现高保真。至 确定真核DNA聚合酶区别对待的机制 在聚合过程中正确和错误的核苷酸之间，我们有 分析了DNA聚合酶-β产生的碱基替换 经典的错误编码和瞬时未对齐机制，使用稳定 状态酶动力学分析。过去一年的一个主要关注点是 一直在继续审查两位推定的 复制DNA聚合酶，阿尔法和德尔塔。我们已经确定， 四个亚基DNA聚合酶α-DNA引物酶复合体的纯化 不同来源的免疫亲和层析法不能更准确 而不是常规纯化的聚合酶。研究以考察 聚合的可加工性和保真度之间的关系 开始了。与波尔阿尔法不同，DNA聚合酶Delta是高度准确的，它 至少在一定程度上是由于核外去核校对。有两种形式的 正在研究的酶是一种可刺激增殖细胞核抗原的形式和不依赖于增殖细胞核抗原的形式。 后一种酶对碱基取代误差的准确度很高， 目前正在进行正向突变测试，以检查错误 专一性。前一种酶正在接受类似的测试，而 增殖细胞核抗原的参与及其对保真度的贡献(如果有的话)在 调查。

Replication and maintenance of the stability of genetic information requires the accurate synthesis of DNA. In animal cells, DNA synthesis is performed by four distinct classes of DNA polymerases, alpha, beta, delta, and gamma. Our objective has been to characterize the accuracy of DNA synthesis by each of these enzymes and to analyze the errors committed by each in an attempt to understand how mutation rates are controlled. Having found that the high fidelity of the mitochondrial replicative DNA polymerase gamma from chick results from exonucleolytic proofreading, we searched for and found similar activity in mammalian gamma polymerases from two sources. This generalizes the discovery of proofreading activity associated with this class of polymerase, and establishes that two of the four classes of higher eukaryotic DNA polymerases achieve high fidelity by a proofreading mechanism. to determine the mechanisms by which eukaryotic DNA polymerases discriminate between correct and incorrect nucleotides during polymerization, we have analyzed base substitutions produced by DNA polymerase-beta by both classical miscoding and transient misalignment mechanisms, using steady state enzyme kinetic analyses. A major focus during the past year has been a continuing examination of the fidelity of the two putative replicative DNA polymerase, alpha and delta. We have established that the four-subunit DNA polymerase alpha-DNA primase complex purified from four different sources by immunoaffinity chromatography is no more accurate than the conventionally purified polymerase. Studies to examine the relationship between the processivity and fidelity of polymerization have begun. Unlike Pol alpha, DNA polymerase Delta is highly accurate, which is at least in part due to exonucleolytic proofreading. Two forms of the enzyme are being studied, a PCNA-stimulable and a PCNA-independent form. The latter enzyme is highly accurate for base substitution errors and is currently being tested in a forward mutation assay to examine error specificity. The former enzyme is being similarly tested, and the involvement of PCNA and its contribution to fidelity, if any, are under investigation.