MECHANISMS OF ULTRAVIOLET LIGHT INDUCED P53 MUTATIONS

我们开发了连接介导的PCR（LMPCR）来绘制 诱变剂诱导的损伤在核苷酸分辨率，并表明，修复 UV诱导的环嘧啶二聚体的速率变化超过十五倍， 核苷酸位置到核苷酸位置。 紫外诱导环丁烷 二聚体，如果没有修复，随后被DNA聚合酶误读， 在突变中，如果它们赋予肿瘤生长优势， 在非黑色素瘤皮肤肿瘤中。 紫外线诱导的主要成分 突变特征是在二嘧啶位点处的C->T转换。 我们表明 环丁烷二聚体修复率沿着p53基因是高度 与频率相关，每个位置呈现为C->T转变 在非黑色素瘤皮肤肿瘤的p53突变数据库中， 内部肿瘤 因此，环丁烷二聚体修复速率的变化沿着 p53基因驱动了一个非常重要的部分的变化， 核苷酸位置间突变率，突变 p53基因在皮肤肿瘤中的突变。 在慢性紫外线照射下， 核苷酸位置达到稳态损伤平衡频率， l当量 发现任何一个核苷酸的修复动力学 位置是一级动力学，我们能够从数学上证明 Leq是急性紫外线剂量乘以 半寿命维修。 因此，L合并了四个顺序中的两个 突变的步骤，并应更高度相关的C->T 转换频率比单独的修复率高。 我们将使用连接介导的PCR来绘制Leq，Laute和修复率。 应用连接介导的PCR检测UV-B辐射的成纤维细胞和角质形成细胞 并确定这些诱变指标中哪一个最能预测C->T 非黑色素瘤皮肤p53突变数据库中的转换频率 肿瘤的 由于环嘧啶二聚体修复率随慢性剂量而变化， 强度（适应性反应）和环境相关剂量是 低剂量时，我们将绘制低UV剂量下的Leq图，以确定相关性 低剂量系数Leq。 为了衡量这一点，我们将增加 通过含有限制性酶切的p53的大小纯化的LMPCR的灵敏度 片段

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.