Genome-wide detection of UV DNA damage by single molecule real time sequencing

描述（由申请人提供）：紫外线辐射是皮肤癌的主要环境原因。然而，尽管已知的病因学影响的紫外线照射，检测初始UV-DNA损伤和随后的突变过程的方法是非常有限的。目前迫切需要一种高通量的方法，可以直接检测UV-DNA损伤的基因组水平在人类细胞。在这里，我们建议开发一种新的方法，基于受损的DNA免疫沉淀（DDIP）富集，然后进行单分子实时（SMRT）DNA测序。DDIP-SMRT序列的开发将对我们直接评估紫外线辐射和其他环境DNA损伤剂对细胞DNA的影响的能力产生重大影响。为了开发DDIP-SMRT序列，我们提出了两个目标。在目标1中，我们将首先通过SMRT DNA测序确定UV诱导的DNA损伤的动力学特征。特别地，我们将推导出由UV辐射形成的环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮光产物（6-4）PPs的聚合酶动力学特征。在目标2中，我们将使用动力学特征直接检测UV暴露的黑素细胞中的CPD和（6-4）PP。来自黑素细胞的DDIP富集的DNA片段将用作两条链的SMRT DNA测序的模板。将优化动力学分析算法，以实现在全基因组水平上灵敏和准确地检测UV-DNA损伤位点。总之，这些目标将改变我们对太阳紫外线辐射（最常见的人类癌症病原体）如何导致皮肤癌的理解，并将促进新的癌症预防策略。一旦开发出来，DDIP-SMRT seq方法可以扩展到研究其他环境致癌物的影响。

DESCRIPTION (provided by applicant): UV radiation is the leading environmental cause of skin cancers. However, despite the known etiological effects of UV exposure, methods for detecting initial UV-DNA damage and the mutational processes that follow are severely limited. There is an urgent need for a high-throughput method that can directly detect UV-DNA damage at the genome level in human cells. Here, we propose to develop a new method based on damaged DNA immunoprecipitation (DDIP) enrichment followed by single- molecule, real-time (SMRT) DNA sequencing. The development of DDIP-SMRT seq will have a major impact on our ability to directly assess the effects of UV-radiation and other environmental DNA damaging agents on cellular DNA. To develop DDIP-SMRT seq, we propose two aims. In aim 1, we will first determine the kinetic signatures of UV-induced DNA damage by SMRT DNA sequencing. In particular, we will derive the polymerase kinetic signatures for cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts (6-4) PPs formed by UV-radiation. In aim 2, we will use the kinetic signatures to directly detect CPDs and (6-4) PPs in UV-exposed melanocytes. The DDIP-enriched DNA fragments from the melanocytes will serve as templates for SMRT DNA sequencing of both strands. The kinetic analysis algorithms will be optimized to enable sensitive and accurate detection of the UV-DNA damage sites at the whole-genome level. Together, these aims will transform our understanding of how solar UV radiation, the most common human cancer etiological agent, leads to skin cancer and will facilitate new cancer-prevention strategies. Once developed, the DDIP-SMRT seq method can be extended to study the effects of other environmental carcinogens.