Deoxyribonucleotide Pool Asymmetry and Genome Variation

对近亲哺乳动物物种间同源基因序列的分析表明，基因组内不同基因的进化速率不同。基因变异率与基因的核苷酸组成有关，特别是与鸟嘌呤+胞嘧啶含量有关。在哺乳动物细胞中，dGTP几乎总是近端DNA前体库中最不丰富的脱氧核糖核苷三磷酸（dNTP）。如果复制DNA聚合酶的dGTP水平处于过饱和状态，复制保真度就会受到影响，这一方面是由于与更丰富的核苷酸竞争插入相反的模板胞嘧啶，另一方面是由于新合成DNA中dGTP上游的校对增加。我们将在Roberts和Kunkel （Proc. Natl）开发的正向突变测定系统中分析体外复制保真度。学会科学。美国法典85,7064（1988）。该系统使用改良的M13噬菌体模板，该模板包含SV40病毒复制源和部分大肠杆菌lacZ基因和控制区。在HeLa细胞提取物复制过程中产生的lac序列靶点突变，可以在显色底物存在的情况下通过空斑测定进行评分。首先，我们将问，当提供近似核内环境的dNTP混合物时，该系统是否产生与等摩尔dNTP混合物所见的突变频率显着不同。然而，关键的测试将是在不改变其编码特性的情况下系统地改变目标DNA序列的GC含量，并询问在不对称dNTP混合物存在的情况下复制时突变频率是否随GC含量的变化而变化。GC含量的变化将涉及化学合成的寡核苷酸取代部分天然lacZ序列。了解与哺乳动物细胞突变有关的参数，以便更好地控制它们，是很重要的。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.