解旋酶RecQL4调控DNA双链断裂损伤修复通路选择的分子机制

DNA double strand break (DSB) repair is mainly done by two pathways: error-free homologous recombination (HR) and error-prone nonhomologous end joining (NHEJ), and for the latter one, both underactivity and overactivity have been shown to contribute to DNA repair infidelity in neoplastic cells. Human helicase RecQL4 has been demonstrated to be a double-edged sword protein, and both mutations and overexpression are involved in tumor progression, and was also shown to participate in HR regulation. However, whether it is involved in NHEJ pathway and further regulates the DNA DSB repair pathway choice are not clear. Our preliminary findings showed that RecQL4 knock-down resulted in a significant reduction in both HR and NHEJ repair activities. Moreover, RecQL4 interacts with Ku80 and ubiquitination E3 ligase RNF8, two critical proteins in NHEJ pathway, and RNF8 silencing led to a significant decrease in RecQL4 ubiquitination level and makedly prolonged recruitment of RecQL4 in DNA DSB sites, suggesting an important role of RecQL4 in regulating NHEJ repair activity. This application aims to elucidate the molecular mechanism of RecQL4 in regulating DNA DSB repair pathway choice, and using the in vitro and in vivo models to define the role of RecQL4 upregulation in affecting genomic stability and subsequent tumor progression. Overall, the findings will form the basis for delineating the role of RecQL4 dysregulation in human disese and provide potential marker for a better design of precaution and intervention approaches.

DNA双链断裂(DSB)修复主要由高保真同源重组(HR)和易错非同源末端连接(NHEJ)两个通路来完成，而且后者过高或过低的活性都与基因组不稳定性相关。解旋酶RecQL4突变缺失或过表达都参与肿瘤进展，而且被证实调控HR修复，但RecQL4是否参与NHEJ并调控DSB修复通路选择目前还不清楚。我们前期结果发现敲低RecQL4导致HR和NHEJ修复活性都明显降低；RecQL4与NHEJ通路蛋白Ku80及泛素化E3连接酶RNF8互作，敲低RNF8可明显降低RecQL4泛素化水平及显著延长RecQL4在DSB损伤位点招募，提示RecQL4对NHEJ起重要调控作用。本申请目标是阐明RecQL4调控NHEJ活性和DSB损伤修复通路选择分子机制；确定其异常过表达是否通过促进NHEJ修复而导致基因组不稳定性和肿瘤易感。相关结果将为阐明RecQL4参与人类疾病发生分子机制提供理论依据和重要分子干预靶标。

DNA双链断裂(DSB)修复主要由高保真同源重组(HR)和易错非同源末端连接(NHEJ)两个通路完成，而后者过高或过低的活性都与基因组不稳定性相关。人类DNA解旋酶RecQL4参与DNA复制和修复，RecQL4的缺失与以基因组不稳定性为特征的常染色体隐性遗传疾病相关，而RecQL4泛素化状态是否对其DNA损伤修复功能至关重要目前尚不清楚。前期研究结果发现敲低RecQL4导致HR和NHEJ修复活性都明显降低；RecQL4与NHEJ通路蛋白Ku80及泛素化E3连接酶RNF8互作，敲低RNF8可明显降低RecQL4泛素化水平及显著延长RecQL4在DSB损伤位点招募，提示RecQL4对NHEJ起重要调控作用。本研究通过系列实验证实RecQL4与RNF8（一种E3泛素连接酶）蛋白直接互作，且共定位于DNA DSB位点。 RecQL4蛋白的K101、K876和K1048位点泛素化受RNF8催化调节。RNF8泛素化修饰RecQL4并促进结合的RecQL4解离；DSBs位点RecQL4突变体的解离延迟并可能抑制下游DSB修复蛋白的募集。通过HR和NHEJ报告系统进一步证实RecQL4敲低后明显降低DNA损伤修复效率，而回补野生型RecQL4可恢复DNA损伤修复能力。本研究还证实RecQL4与WRAP3β蛋白互作，敲低WRAP3β后细胞核RNF8在DSBs位点的募集减少，并造成RecQL4在DSBs位点滞留时间明显延长；同时RecQL4与RNF8的互作也随WRAP3β 蛋白水平升高而增强。上述结果表明RNF8介导的泛素化事件在RecQL4参与的DSB修复中发挥重要作用，为阐明RecQL4参与人类疾病发生分子机制提供理论依据。此外，本研究还证实了RecQL4对线粒体能量代谢具有促进作用，而SIRT4对RecQL4的去乙酰化会抑制线粒体能量代谢，该结果对线粒体疾病发病机制的研究具有重要意义。