Deoxyribonucleotide Pool Asymmetry and Genome Variation

近缘种间同源基因序列分析 哺乳动物的基因组中的不同基因 以不同的速度进化。 基因变异率与 基因的核苷酸组成，特别是， 鸟嘌呤+胞嘧啶含量。 在哺乳动物细胞中， 总是最少的脱氧核糖核苷三磷酸 在近端DNA前体池中的dNTP。 如果dGTP水平 复制型DNA聚合酶的亚饱和，复制保真度 会受到影响，这两个因素都是与更丰富的核苷酸竞争， 对于插入相反的模板胞嘧啶，并通过增加 在新合成的DNA中从dGMP上游校正。 我们将 在正向突变试验中分析体外复制保真度 由Roberts和Kunkel开发的系统（Proc.Natl. Acad. Sci.美国 85，7064（1988）中所述。 该系统使用修饰的M13噬菌体模板 其包含SV40病毒复制起点和一部分 E. colilacZ基因和控制区。 紫胶中的突变 序列靶标，在HeLa细胞提取物复制期间产生， 在显色剂存在下通过噬斑测定进行评分 衬底 首先，我们要问的是，这个系统在提供时， 用近似核内环境的dNTP混合物， 产生的突变频率与我们所看到的 与等摩尔的dNTP混合。 然而，关键的测试将 系统地改变靶DNA序列的GC含量 而不改变其编码特性，并询问突变是否 频率变化的GC含量的函数时，复制在 不对称dNTP混合物的存在。 GC含量变化 将涉及取代化学合成的 天然lacZ序列部分的寡核苷酸。 %%% 重要的是要知道与之相关的参数。 哺乳动物细胞中的突变，以便更好地控制它们。 的 这项研究的信息将提高我们这样做的能力。

Analysis of homologous gene sequences among closely related mammalian species reveals that different genes within a genome evolve at different rates. The rate of gene variation is related to the nucleotide composition of the gene, specifically, to the guanine+cytosine content. In mammalian cells dGTP is almost invariably the least abundant deoxyribonucleoside triphosphate (dNTP) in the proximal DNA precursor pool. If dGTP levels are subsaturating for replicative DNA polymerases, replication fidelity can be affected, both by competition with more abundant nucleotides for insertion opposite template cytosines, and by increased proofreading upstream from dGMP in newly synthesized DNA. We will analyze replication fidelity in vitro in a forward mutation assay system developed by Roberts and Kunkel (Proc. Natl. Acad. Sci. USA 85, 7064 (1988). This system uses a modified M13 phage template that contains an SV40 viral replication origin and a portion of the E. coli lacZ gene and control region. Mutations in the lac sequence target, arising during replication by a HeLa cell extract, are scored by a plaque assay in the presence of a chromogenic substrate. First we will ask whether this system, when provided with dNTP mixtures that approximate the intranuclear environment, yields mutation frequencies significantly different from those seen with an equimolar dNTP mixture. The critical test, however, will be to systematically vary the GC content of the target DNA sequence without changing its coding properties, and to ask whether mutation frequencies vary as a function of GC content when replicated in the presence of asymmetric dNTP mixtures. The GC content variation will involve substitution of chemically synthesized oligonucleotides for portions of the natural lacZ sequence. %%% It is important to know the parameters which are associated with mutations in mammalian cells in order to better control them. The information from this study will advance our ability to do so.