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

摘要： 暴露于太阳紫外线（UV）光会造成DNA损伤，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环丁烷嘧啶二聚体（CPD） 和在二嘧啶序列处的6 - 4个光产物（6 - 4PP）（即，TT、TC、CT和CC）。脱氨基 这些损伤或诱变旁路中的胞苷导致其频繁转化为突变。因此，UV- 暴露的细胞和皮肤癌主要由二嘧啶序列中的C至T和CC至TT取代所控制 共同构成了紫外线突变特征令人惊讶的是，许多驱动突变，有助于 黑色素瘤进展具有序列背景和取代特征，这些特征不符合 典型的紫外线变异特征例如，最常见的黑色素瘤驱动突变BRAF V600E， 涉及在GTG序列背景下T至A的取代。这种突变特征的差异， 大多数紫外线诱导的突变和黑色素瘤驱动突变导致了这样的假设： 通过诱导突变以外的机制引起黑色素瘤。然而，我们最近提供了 全基因组测序中罕见的非典型紫外线损伤引起突变的实验证据 酵母和生物信息学证据表明人类临床皮肤癌中存在类似病变。这些病变很可能 在TA、CA和AC二核苷酸处形成的大体积光产物。引人注目的是，与这些基因相关的突变 非典型UV光产物具有与黑素瘤中许多复发性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起着重要作用。的目的 一项建议是更好地确定非典型紫外线光产物的特征及其对癌症的影响 进展在目的I中，我们将利用酵母中表达的CPD和6 - 4PP光解酶来分配UV诱导的 突变类别与其相应的病变类型，以及评估生理UVB光的能力， 在酵母和人类细胞中诱导与非典型UV光产物相关的突变类型。在Aim II中，我们将 使用新的高通量测序分析非典型TA光产物的全基因组分布 方法，称为UVDE-seq。我们还将表征推定的CA和AC光产物的形成， 它们对致癌BRAF突变的贡献。最后，Aim III将确定参与DNA聚合酶的作用。 TA和AC光产物的无错误和易出错旁路。成功实现这些目标将 为皮肤癌的分子原因提供了新的见解，从而定义了紫外线的新范式。 突变，可能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.