ANALYSIS OF MOLECULAR MECHANISMS OF MUTAGENESIS WITH DEFINED COMPONENTS

遗传信息稳定性的复制和维持 需要精确的DNA合成。在动物细胞中，DNA合成是 由四种不同的DNA聚合酶，α，β， δ和γ。我们的目标是描述 通过这些酶中的每一种合成DNA，并分析错误 试图了解突变率是如何 控制。我们发现线粒体的高保真度 鸡的复制性DNA聚合酶γ由核酸外切引起 校对，我们在哺乳动物中寻找并发现了类似的活性， 两种来源的γ聚合酶。这概括了 与这类聚合酶相关的校对活性，和 确定了四种高等真核DNA中的两种 聚合酶通过校对机制实现高保真度。到 确定真核生物DNA聚合酶区分的机制 在聚合过程中正确和不正确的核苷酸之间， 分析了DNA聚合酶β产生的碱基替换， 经典的错误编码和瞬态错位机制，使用稳定 进行酶动力学分析。过去一年的一个主要重点是 这是一个持续的审查的忠诚度的两个假定 复制DNA聚合酶α和δ我们已经确定， 四亚基DNA聚合酶α-DNA引发酶复合物， 不同来源的免疫亲和色谱法是没有更准确 比常规纯化的聚合酶。研究以审查 聚合的持续性和保真度之间的关系具有 开始.与Pol alpha不同，DNA聚合酶Delta是高度精确的， 至少部分是由于核酸外切校正。两种形式的 一种PCNA刺激型和非PCNA依赖型。 后一种酶对于碱基置换错误是高度准确的， 目前正在进行正向突变试验， 的特异性前一种酶正在进行类似的测试， PCNA的参与及其对保真度的贡献，如果有的话， 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.