癌细胞基因组中尿嘧啶修复引发突变的分子机制

Uracil is one of the major abnormal bases in genomic DNA, and its proper repair is closely related to the stability of genome; whereas the DNA repair system in cancer cells is often aberrant, which could result in genome instability. By using a in-vitro generated plasmid vector, our previous work has shown that the repair of uracil can cause mutations in flanking DNA sequences, in which the cytidine deaminase APOBEC mediates the mutagenic process. Based on the hypermutations at TpC dinucleotides in genomic DNA and the unusual over-expression of APOBEC in cancer cells, we propose to further study the molecular mechanism of uracil repair-induced mutagenesis in cancer genomes and would mainly focus on three aspects: 1) constructing the experimental system to induce uracils at targeted genomic sites, 2) revealing whether APOBEC mediates mutagenesis during the repair of uracils in cancer genomic DNA, 3) detecting uracil distribution, APOBEC expression and genomic mutations in clinic cancer tissues. The proposed study aims to reveal novel molecular mechanisms of the mutagenesis in cancer genome, which would provide principles to develop new therapeutic strategies and medicines for the relevant cancers.

尿嘧啶作为基因组DNA中的主要异常碱基之一，其正确修复与基因组的稳定性密切相关；而癌细胞多发DNA修复异常，易导致基因组的不稳定性。我们前期工作利用体外构建含尿嘧啶的质粒载体研究发现，尿嘧啶的修复过程可引发旁侧质粒DNA序列突变，且是由识别TpC位点的胞嘧啶脱氨酶APOBEC介导发生。本申请项目依据癌细胞基因组中TpC位点存在高频突变以及APOBEC异常性高表达等线索，拟进一步探索癌细胞基因组中尿嘧啶修复引发突变的分子机制。主要开展：⑴基因组DNA中定点产生尿嘧啶的实验体系构建；⑵APOBEC是否在癌细胞基因组DNA尿嘧啶修复过程中介导突变发生；⑶癌症临床样本中的基因组DNA尿嘧啶产生、APOBEC表达及其突变检测等研究。旨在揭示癌细胞基因组突变的新型分子机制，为相关癌症的诊治策略研究和药物研发等提供理论依据。

尿嘧啶作为基因组DNA中的主要异常碱基之一，其正确修复与基因组的稳定性密切相关；而癌细胞多发DNA修复异常，易导致基因组的不稳定性。我们前期工作利用体外构建含尿嘧啶的质粒载体研究发现，尿嘧啶的修复过程可引发旁侧质粒DNA序列突变，且是由识别TpC位点的胞嘧啶脱氨酶APOBEC介导发生。本项目研究中依据癌细胞基因组中TpC位点存在高频突变以及APOBEC异常性高表达等线索，通过筛选和研究改造APOBEC构建了不激活DNA损伤响应通路的碱基编辑器（BEACON）和新型高精准变形式碱基编辑系统（tBE），并将这些构建的碱基编辑器转入到人源癌细胞系中，比较他们在特定位点引发突变的程度，揭示了APOBEC在癌细胞基因组DNA修复过程中介导突变发生有直接的作用，为探索癌细胞基因组中DNA修复引发突变提供了新工具，也为相关癌症的诊治策略研究和药物研发等提供了理论依据。