DNA解旋酶RECQL在哺乳动物DNA双链断裂修复中的作用及机制研究

RECQL, encoding a member of the RECQ helicase family, is a recently identified familial breast cancer susceptibility gene. More RECQL variants of unknown clinical significance (VUS) are found with the screening of RECQL mutations in familial breast cancer. As RECQL-deficient cells exhibit significant genomic instability, which is largely associated with defects in DNA double strand break (DSB) repair, it becomes more important to understand whether and how RECQL mutations promotes genomic instability and thus the susceptibility to cancer. Preliminary work has shown that the RECQL deficiency causes defects in two major DSB repair pathways: homologous recombination (HR), particularly the HR pathway coupled with restart of stalled replication forks, and non-homologous end-joining (NHEJ). Together with the biochemical activities of RECQL in DNA binding, DNA unwinding and branch migration of repair intermediates, we hypothesize that RECQL may recognize and bind specific DNA configurations and use its helicase activity to regulate HR and NHEJ, helping to maintain the integrity of the genomes in cells. To test this hypothesis, we propose to: 1) Identify the role of RECQL in HR repair of two-ended DSB independently of DNA replication; 2) Dissect the role of RECQL in HR repair of DSBs converted by DNA replication from single-strand nicks; 3) Study the role of RECQL in NHEJ repair of DSBs; 4) Examine clinical relevance of the RECQL deficiency in cancer. Our objectives are to elucidate the molecular mechanisms by which RECQL control HR and NHEJ and to establish molecular connection between RECQL’s function and phenotypes of cancer cells. We hope in the long term to fully understand the initiation and development of RECQL-deficient human diseases, which include RECQL-deficient familial breast cancer, thus providing guidance in prevention, diagnosis and treatment of these diseases.

RECQL是新近发现的一个家族性乳腺癌易感基因，其缺陷导致明显的基因组不稳定性。前期工作显示RECQL缺陷引起细胞内DNA双链断裂（DSB）修复途径同源重组（HR）和非同源末端连接（NHEJ）缺陷，特别是与DNA复制相关的HR。结合RECQL的生化活性，我们推测，RECQL利用其识别特定DNA构象的能力及解旋酶活性，参与调节HR和NHEJ，维护基因组稳定性。因此，通过研究哺乳动物细胞内RECQL对HR和NHEJ的调控，包括与DNA复制偶联的HR和NHEJ，揭示RECQL的功能及其分子机制，建立细胞和肿瘤内RECQL缺陷基因型与表型之间的分子关联。同时，随着家族性乳腺癌RECQL突变筛查得以重视，更多临床意义不明的RECQL突变被发现。通过测试这些突变可能的DSB修复功能缺陷及其对特定药物（比如PARP抑制剂）的敏感性，不仅有助于理解这类疾病的发生发展，而且可以帮助指导其预防、诊断和治疗。

RECQL及其相关因子（比如BRCA1和BLM等）的突变与恶性肿瘤的发生发展密切相关。这些因子也被认为参与调控DNA双链断裂（DSB）修复，包括同源重组（HDR）和非同源末端连接（NHEJ）修复途径，但其中的作用及机制还有诸多未知。为了阐明RECQL及相关因子在复制偶联单末端DSB和传统的双末端DSB修复中的异同及其对肿瘤基因组瘢痕的影响，我们联合CRISPR/Cas9的靶点单链和双链切割能力及其靶点滞留效应，分析了这些因子在DSB修复中的作用与机制，产生如下结果和发现：.1）CRISPR靶点滞留调控DSB修复途径选择，滞留时间长的靶点有更高概率与DNA复制叉碰撞，产生经典型NHEJ（c-NHEJ）不易参与的末端，偏向HDR选择，而滞留短的靶点易产生游离的末端，偏向于c-NHEJ。.2）c-NHEJ小分子抑制剂常用于提升HDR介导的基因编辑，但也加重脱靶效应。为解决这个问题，我们建立了基于dCas9的c-NHEJ定点抑制策略，可以定点提升HDR介导的基因编辑效率，还可以避免脱靶效应恶化。.3）借助CRISPR的单链切割和靶点滞留效应，建立DNA复制偶联的单末端DSB的修复报告系统，发现RECQL在DNA复制偶联单末端DSB和传统的双末端DSB修复中作用有限。.4）建立了DNA重复制诱导DSB修复的报告系统，在此基础上发现BLM抑制重复制偶联的DSB的生成或重复制偶联的HDR。.5）BRCA1缺陷肿瘤突变模式的一个主要诱因是复制偶联单末端DSB的产生及修复异常，而不是传统认为的双末端DSB。.该项目不仅阐明了CRISPR/Cas9的靶点滞留对DSB修复途径选择的影响，还揭示了RECQL等因子在DNA复制偶联单末端DSB修复、传统的双末端DSB修复和DNA重复制诱导HDR中作用，以及对肿瘤基因组瘢痕产生的贡献。这不仅将推动DSB修复机制的全面了解，也将有助于理解相关疾病的发生和演化。

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