Mechanisms of mutagenesis in " hot spots" causing cancer

描述(申请人提供)：这些研究的长期目标是使用两个微生物模型系统来确定压力引发的基因转录和超螺旋增加突变率的机制，特别是在激活的基因中。这些突变发生在已知致癌的相同类型的二级结构中。我们实验室最近的证据表明，在微生物和人类中，转录都驱动DNA超螺旋，从而产生茎环结构(SLS)，不成对的碱基容易发生突变。应激源也可以直接影响负超螺旋，导致更多的SLS和更高的突变率。在我们的大肠杆菌模型系统中，我们将确定压力对转录、超螺旋和突变频率的影响，获得在转录和非转录链中预测的SLS存在的证据，并修改SLS中的序列以检查预测的SLS对突变率的影响。 在我们的酿酒酵母模型系统中，我们将检测各种环境应激源对p53癌基因特定高突变碱基逆转的影响。利用DNA折叠程序和一种新的计算机算法，我们定位了DNA SLS中的致癌碱基，并成功地预测了它们的突变指数(Mis)。通过酵母-质粒模型，我们希望验证我们预测突变频率的方法，并促进我们对导致突变的变量的理解。例如，我们将比较同一SLS中P53高突变碱基和对照碱基的已知结构、错位和正向(野生型到突变型)突变频率与反向突变(突变型到野生型)的突变频率。我们将比较转录、超螺旋和应激源对SLS中不同错失和位置不同碱基突变频率的影响：可以比较已知致癌物对SLS同一脆弱位置不同碱基的返回率的影响，以阐明1)碱基本身与2)碱基位置在确定突变频率方面的相对重要性。这些研究应该为突变和致癌的机制提供新的见解。

DESCRIPTION (provided by applicant): The long-term goal of these investigations is to use two microbial model systems to determine the mechanisms by which gene transcription and supercoiling triggered by stress increase mutation rates specifically in the activated genes. These mutations occur in the same kinds of secondary structures known to cause cancer. Recent evidence from our laboratory suggests that, in both microbes and man, transcription drives DNA supercoiling which creates stem-loop structures (SLS) with unpaired bases vulnerable to mutations. Stressors can also affect negative supercoiling directly, resulting in more SLS and higher mutation rates. In our E. coli model system we will determine the effects of stress on transcription, supercoiling, and mutation frequency, obtain evidence for the existence of predicted SLS in both transcribed and non-transcribed strands, and modify sequences in SLS to examine predicted effects on mutation rates. In our S. cerevisiae model system, we will examine the effects of various kinds of environmental stressors on reversion rates of specific hypermutable bases of the p53 cancer gene. Using a DNA folding program and a new computer algorithm we have localized cancer-causing bases in DNA SLS and successfully predicted their mutability indexes (MIs). With the yeast-plasmid model we hope to verify our method for predicting mutation frequencies and advance our understanding of the variables that cause mutations. For example, we will compare known structures, MIs and forward (wild to mutant) mutation frequencies inp53 hypermutable and control bases to those of the backward (mutant to wild) mutations, in the same SLS. We will compare the effects of transcription, supercoiling, and stressors on mutation frequencies of bases that differ in MIs and locations in SLS: The effects of known carcinogens on reversion rates of different bases in the same vulnerable position of a SLS can be compared, to clarify the relative importance of 1) the base per se, vs 2) the location of the base in determining mutation frequencies. These investigations should provide new insights into the mechanisms of mutagenesis and carcinogenesis.