ERROR CORRECTION IN DNA SYNTHESIS--A BIOCHEMICAL STUDY

突变在生物体中扮演着积极和消极的核心角色。 有机体 单碱基替换是最简单的一类 突变，但它们可能会产生深远的生物学后果。 他们可以 作为进化的驱动力。 也有许多 一个碱基的改变就能导致癌症的例子 人类基因组中的一对。 遗传性疾病也是如此， 镰状细胞性贫血和莱-尼二氏综合征 众所周知，自 最早的致突变研究是在阐明 突变以非随机方式沿着 基因组 沿着DNA的某些沿着位点，称为“热点”， 远高于平均突变频率，而其他网站，“冷” 斑点”，以显著降低的速率突变。 的焦点 这项拨款提案是为了研究冷热碱基替代 分子水平上的斑点。 众所周知，近端甚至 远端碱基序列可以强烈地影响在给定的位置处的碱基替换。 DNA位点。 我们提出最近邻碱基堆积力 调节DNA聚合酶的核苷酸插入的保真度， 延伸含有单碱基错配的DNA的效率。 它有 还提出DNA的相对稳定性（即，的 A-T/G-C碱基对的比率）可以深刻地影响 在复制叉处，通过校对核酸外切酶进行纠错。 我们正在利用聚丙烯酰胺凝胶试验来测试聚合酶模型 忠实度作为基本上下文的函数。 每个保真度组件： 可以测量核苷酸插入、延伸和/或切除 独立地。 我们将确定是否有有趣的相关性， 最近邻核苷酸和酶之间的早期观察 歧视机制可以通过获得额外的 独立数据。 除了涉及正常的碱基置换外， 碱基错配，有生物学意义的病变，可以影响 DNA的模板特性 失去一个碱基， 非指导性（碱基）损伤，而碱基烷基化可以改变碱基 配对特异性 已知非碱性位点和烷基化碱基是 高度有害的损伤，导致复制阻断，诱变， 和致癌作用。 我们打算用凝胶保真试验来分析 基础语境对插入、延伸和校对的影响. 选择DNA上的病变部位。 最后，等位基因选择性扩增 使用聚合酶链反应已经成为一种重要的方法， 鉴定克隆DNA中人类起源的突变。 我们的实验 基础环境对经过损伤的延伸的影响将具有实际意义 在这些实验的设计和分析中的应用。

Mutations play both a positive and negative central role in an living organisms. Single base substitutions are the simplest class of mutations, yet they can have profound biological consequences. They can act as a driving force governing evolution. There are also numerous examples of cancers that can result from an alteration of just one base pair in the human genome. The same is true for inherited diseases such as sickle cell anemia and Lesch-Nyhan syndrome. It is well known since the earliest mutagenesis studies following the elucidation of the structure of DNA, that mutations occur in a nonrandom fashion along the genome. Certain sites along DNA, referred to as "hot spots", exhibit much higher than average mutation frequencies, while other sites, "cold spots", mutate at a significantly diminished rate. The focal point of this grant proposal is to investigate base substitution hot and cold spots at a molecular level. It is well known that proximal and even distal base sequences can strongly affect base substitutions at a given DNA locus. We have proposed that nearest-neighbor base stacking forces modulate the fidelity of nucleotide insertion of DNA polymerase and the efficiency of elongating DNA containing a single base mismatch. It has also been suggested that the relative stability of the DNA (i.e., the ratio of A-T/G-C base pairs) can profoundly affect the efficiency of error correction, by proofreading exonucleases, at the replication fork. We are utilizing a polyacrylamide gel assay to test models of polymerase fidelity as a function of base context. Each fidelity component: nucleotide insertion, extension and/or excision can be measured independently. We will determine whether interesting correlations we had earlier observed between nearest neighbor nucleotides and enzyme discrimination mechanisms can be supported by obtaining additional independent data. In addition to base substitutions involving normal base mispairs, there are biologically significant lesions that can affect the templating properties of DNA. The loss of a base results in a noninstructional (abasic) lesion, while base alkyations can alter base pairing specificities. Abasic sites and alkyated bases are known to be highly deleterious lesions, causing replication blockage, mutagenesis, and carcinogenesis. We intend to use the gel fidelity assay to analyze the effects of base context on insertion, extension and proofreading at selected lesion sites on DNA. Finally, allele selective amplification using the polymerase chain reaction has become an important method to identify mutations of human origin in cloned DNA. Our experiments on the effects of base context on extension past a lesion will have practical application in the design and analysis of these experiments.