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

通过单分子实时测序全基因组检测 UV DNA 损伤

基本信息

批准号：
8807049
负责人：
ANEEL K. AGGARWAL
金额：
$ 25.43万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8807049
关键词：
Algorithms Architecture Base Sequence Carcinogens Cataloging Catalogs Cells Chromatin Cutaneous Melanoma DNA DNA Damage DNA Sequence DNA lesion DNA-Directed DNA Polymerase Detection Development Environmental Carcinogens Etiology Exposure to Genetic Genome Genomic DNA Genomics Goals Hot Spot Human Immunoprecipitation Individual Kinetics Lesion Ligation Location Malignant Neoplasms Maps Mediating Methods Monitor Mutation Nucleotides Polymerase Prevention strategy Process Pyrimidine Pyrimidine Dimers Reporting Resolution Single-Stranded DNA Site Skin Cancer Skin Carcinoma Specificity Stretching Techniques Time UV Radiation Exposure UV induced UV induced DNA damage Ultraviolet Rays United States base cancer diagnosis cancer prevention genome-wide melanocyte melanoma novel public health relevance research study single molecule ultraviolet damage

项目摘要

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.

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，尽管紫外线暴露具有已知的病因影响，但检测初始UV-DNA损伤和随后的突变过程的方法受到严重限制。迫切需要一种高通量方法，该方法可以直接检测人类细胞中基因组水平的UV-DNA损伤。在这里，我们建议开发一种基于受损的DNA免疫沉淀（DDIP）富集的新方法，然后是单分子实时（SMRT）DNA测序。 DDIP-SMRT SEQ的发展将对我们直接评估UV辐射和其他环境DNA损害剂对细胞DNA的影响的能力产生重大影响。为了开发DDIP-SMRT SEQ，我们提出了两个目标。在AIM 1中，我们将首先通过SMRT DNA测序来确定紫外线诱导的DNA损伤的动力学特征。特别是，我们将推导环丁烷嘧啶二聚体（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AIM 2中，我们将使用动力学特征直接检测紫外线暴露的黑素细胞中的CPD和（6-4）PPS。来自黑素细胞的富含DDIP的DNA片段将用作两条链的SMRT DNA测序的模板。动力学分析算法将被优化，以实现整个基因组水平上的UV-DNA损伤位点的敏感和准确检测。这些目标共同改变了我们对太阳紫外线辐射（最常见的人类癌症病因剂）如何导致皮肤癌的理解，并将促进新的预防癌症预防策略。开发后，可以扩展DDIP-SMRT SEQ方法以研究其他环境致癌物的影响。