Genome-wide analysis of the formation and mutagenesis of atypical UV photoproducts in skin cancer

摘要： 暴露在太阳紫外线(UV)光下会造成DNA损伤，从而诱导高水平的体细胞突变 人类皮肤癌，如黑色素瘤。紫外线最常见的原因是环丁烷嘧啶二聚体(CPDS) 以及6-4个二嘧啶序列(即TT、TC、CT和CC)的光产物(6-4pps)。脱氨基 这些病变或诱变旁路中的胞苷会导致它们频繁地转化为突变。因此，紫外线- 暴露的细胞和皮肤癌主要由双嘧啶序列中的C到T和CC到TT取代 它们共同构成了一个紫外线突变签名。令人惊讶的是，许多导致 黑色素瘤进展的序列背景和替换特征不符合 典型的紫外线突变签名。例如，最常见的黑色素瘤驱动基因突变BRAF V600E， 在GTG序列上下文中涉及T到A的替换。这一突变特征的差异 大多数紫外线诱导的突变和黑色素瘤驱动因素的突变导致了紫外线诱导的假设 黑色素瘤是通过诱导突变以外的机制发生的。然而，我们最近提供了 全基因组测序中罕见的非典型性紫外线损伤引起突变的实验证据 酵母菌和生物信息学证据表明人类临床皮肤癌存在类似的损害。这些损伤很可能是 在TA、CA和AC二核苷酸上形成的大块光产物。令人惊讶的是，与这些基因相关的突变 非典型紫外线光产物具有与黑色素瘤中许多反复发生的驱动突变相同的特征， 这表明，这些罕见的病变可能在导致皮肤癌方面发挥了重要作用。这样做的目的是 建议更好地定义非典型紫外光产品的特性及其对癌症的贡献 进步。在目标I中，我们将利用在酵母中表达的CPD和6-4PP光解酶来分配紫外线诱导的 以及评估生理UVB光对其损伤类型的能力 在酵母和人类细胞中诱导与非典型紫外光产物相关的突变类别。在AIM II中，我们将 用一种新的高通量测序方法分析非典型TA光产物的全基因组分布 方法，称为UVDE-seq。我们还将表征假定的CA和AC光产物的形成以及 它们对致癌的BRAF突变的贡献。最后，Aim III将鉴定参与 TA和AC感光产品的无差错和易出错旁路。这些目标的成功实现将 为皮肤癌的分子病因提供新的见解，从而定义紫外线的新范式 突变可以潜在地解释急性紫外线暴露与 黑色素瘤发病率增加。

Abstract: Exposure to solar ultraviolet (UV) light creates DNA damage that induces high levels of somatic mutations in human skin cancers like melanoma. UV light most commonly causes cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) at dipyrimidine sequences (i.e., TT, TC, CT, and CC). Deamination of cytidines in these lesions or mutagenic bypass leads to their frequent conversion to mutations. As a result, UV- exposed cells and skin cancers are dominated by C to T and CC to TT substitutions in dipyrimidine sequences that collectively constitute a UV mutation signature. Surprisingly, many driver mutations that contribute to melanoma progression have sequence context and substitution characteristics that do not conform to the canonical UV mutation signature. For example, the most common melanoma driver mutation, BRAF V600E, involves a T to A substitution in a GTG sequence context. This difference in mutation characteristics between most UV-induced mutations and melanoma driver mutations has led to the hypothesis that UV light induces melanomagenesis by mechanisms other than the induction of mutations. However, we have recently provided experimental evidence of mutations caused by rare, non-canonical UV lesions in whole genome sequenced yeast and bioinformatics evidence of similar lesions in human clinical skin cancers. These lesions are likely bulky photoproducts formed at TA, CA, and AC dinucleotides. Strikingly, the mutations associated with these atypical UV photoproducts have identical characteristics to many recurrent driver mutations in melanoma, suggesting that these rare lesions may play a significant role in causing skin cancer. The objective of this proposal is to better define the characteristics of atypical UV photoproducts and their contribution to cancer progression. In Aim I, we will utilize CPD and 6-4PP photolyases expressed in yeast to assign UV-induced mutation classes to their corresponding lesion types as well as assess the ability of physiological UVB light to induce mutation classes associated with atypical UV photoproducts in yeast and human cells. In Aim II, we will analyze the genome-wide distribution of atypical TA photoproducts using a novel high throughput sequencing method, called UVDE-seq. We will also characterize the formation of putative CA and AC photoproducts and their contribution to oncogenic BRAF mutations. Finally, Aim III will identify DNA polymerases involved in the error-free and error-prone bypass of TA and AC photoproducts. Successful completion of these aims will provide new insights into the molecular causes of skin cancer, and thereby define a new paradigm of UV mutagenesis that could potentially explain the epidemiological association of acute UV exposure with increased melanoma incidence.