MECHANISMS OF ULTRAVIOLET LIGHT INDUCED P53 MUTATIONS

我们开发了连接介导的聚合酶链式反应(LMPCR)来定位 诱变剂在核苷酸解析时引起的损伤并显示修复 紫外光诱导的环嘧啶二聚体的速率从 核苷酸位置到核苷酸位置。紫外光诱导环丁烷 二聚体，如果不修复，随后被DNA聚合酶结果误读 在突变中，如果它们赋予致癌生长优势，似乎 在非黑色素瘤皮肤肿瘤中。紫外线诱导辐射的主要成分 突变的特征是二嘧啶的C-&T转变。我们展示了 顺着p53基因的环丁烷二聚体修复率很高 与频率相关，每个位置显示为C->T转变 在非黑色素瘤皮肤肿瘤的P53突变数据库中，但对于 内脏肿瘤。因此，环丁烷二聚体修复率的变化 P53基因驱动了非常重要的一部分变异 核苷酸位置对核苷酸位置的突变率，突变 皮肤肿瘤中P53突变的谱。对于慢性紫外线剂量，每个 核苷酸位置达到稳态损伤平衡频率， LEQ。发现任何一个核苷酸的修复动力学 位置是一级动力学，我们能够用数学表示 LEQ是急性紫外线剂量后易受损害的乘以 维修的半衰期。因此，L将四个序列中的两个合并在一起 突变的步骤，应该与C&GT；T更高度相关 转换频率比单独修复率要高。 我们将使用连接介导的聚合酶链式反应来定位LEQ、L急性和修复率。 UV-B辐射的成纤维细胞和角质形成细胞连接介导的聚合酶链式反应 并确定这些突变指标中哪一项最能预测C&T；T 非黑色素瘤皮肤的P53突变数据库中的转换频率 肿瘤。由于环嘧啶二聚体修复率随慢性剂量不同而不同 强度(适应性反应)和与环境相关的剂量是 在低紫外线剂量下，我们将映射LEQ以确定相关性 低剂量LEQ的系数。为了衡量这一点，我们将增加 含限制性内切酶的P53大小纯化对LMPCR的敏感性 碎片。

We developed ligation-mediated PCR (LMPCR) to map the frequency of mutagen-induced damage at nucleotide resolution and showed that the repair rate of UV-induced cyclopyrimidine dimers varies over fifteen fold from nucleotide position to nucleotide position. UV-induced cyclobutane dimers, if not repaired and subsequently misread by DNA polymerase result in mutations which, if they confer a tumorigenic growth advantage, appear in non-melanoma skin tumors. The major component of the UV-induced mutational signature is C->T transitions at dipyrimidine sites. We show that cyclobutane dimer repair rates along the p53 gene are highly correlated with the frequency each position appears as a C->T transition in the p53 mutation data base for non-melanoma skin tumors, but not for internal tumors. Thus, variation in cyclobutane dimer repair rate along the p53 gene drives a very significant fraction of the variation of the nucleotide position to nucleotide position mutation rate, mutational spectrum, of p53 mutations in skin tumors. With chronic UV doses, each nucleotide position reaches a steady state lesion equilibrium frequency, Leq. On finding that the kinetics of repair at any one nucleotide position are first order kinetics, we were able to show mathematically that Leq is the product of ease of damage after an acute UV dose times the half life for repair. As such, L incorporates two of the four sequential steps of mutagenesis and should be more highly correlated with C->T transition frequency than is repair rate alone. We will use ligation-mediated PCR to map Leq, Lacute, and repair rate in UV-B irradiated fibroblasts and keratinocytes using ligation-mediated PCR and determine which of these mutagenesis metrics best predicts the C->T transition frequency in the p53 mutation data base for non-melanoma skin tumors. Since cyclopyrimidine dimer repair rates vary with chronic dose strength (the adaptive response) and the environmentally relevant dose is a low one, we will map Leq at low UV doses to determine the correlation coefficient of the low dose Leq. To measure this, we will increase the sensitivity of LMPCR by size purification of p53 containing restriction fragments.