衰老相关蛋白SIRT6调控基因组稳定性分子机制的研究

Maintenance of genomic stability is of great importance to prevent cells from entering senescent state. Emerging evidence indicates that SIRT6, a mammalian homolog of Sir2 that extends lifespan in various orgainisms including yeast, nematode and fruit fly, participates in suppressing genomic instabilty, helping improve mouse lifespan. We created sensitive and quantitative assays to measure efficiency of different types of DNA repair including DNA double strand break repair (DSB repair) and nucleotide excision repair (NER) based on green fluorescent protein. With these powerful tools, we found SIRT6 stimulates DNA DSB repair by mono-ADP-ribosylating PARP1, an important repair protein involved in various types of DNA repair. In addition, we also found that SIRT6 enhances NER, but it is independent of PARP1. Moreover, our data shows that SIRT6 is modified by Sumo3, leading to the improvement of SIRT6 stability. To further elucidate molecular mechanisms of regulating genomic stability by SIRT6, based on our preliminary data, we plan to study the following projects. Aim 1: To further investigate roles of SIRT6 in regulating DNA DSB repair, particularly classical nonhomologous end joining (C-NHEJ) pathway. Aim 2: To elucidate the molecular mechanisms of regulating NER by SIRT6. We will mainly focus on identifying new candidate proteins which mediate the regulation of NER by SIRT6. Aim 3: To examine roles of SIRT6 sumoylation in maintaining genomic stablity. Aim 4: To test functional roles of SIRT6 in DNA repair using in vivo mouse models.

提高基因组稳定性对于延缓细胞衰老十分重要。作为哺乳动物SIRTUIN家族的一员，SIRT6是能延长不同低等生物寿命的Sir2的同源基因。证据显示SIRT6通过调控基因组稳定性延缓小鼠衰老。我们构建了检测DNA双链断裂修复和核苷酸切除修复的模型，并利用它们证明在人正常细胞中SIRT6不仅通过单ADP核糖化PARP1提高DNA双链断裂修复效率，也通过不依赖于PARP1的通路促进核苷酸切除修复。此外，小泛素修饰蛋白Sumo3能够修饰SIRT6并提高其稳定性，但其是否参与调控基因组稳定性尚未被研究。为了深入阐明SIRT6调控基因组稳定性的分子机制，我们将在细胞水平上进一步研究SIRT6调控DNA双链断裂修复和核苷酸切除修复的机制，探索SIRT6翻译后修饰如小泛素化对基因组稳定性调控的影响，并利用已构建完善的研究DNA修复的小鼠模型在动物水平上探索SIRT6调控上述两条通路的机制。

衰老相关蛋白SIRT6与基因组稳定性关系紧密，其缺陷可导致基因组不稳定进而诱发衰老，但是领域内对其研究主要集中于其在DNA双链断裂损伤修复中的同源重组修复通路以及替代性非同源末端连接两条通路中的功能。通过该项目资助，我们进一步发现了SIRT6在其它类型DNA损伤修复通路中的功能。我们发现SIRT6通过修饰PARP1的第521的赖氨酸进而激活PARP1的活性从而促进碱基切除修复（BER）通路，而在年老人来源的细胞中SIRT6的表达逐步下调导致其调控的BER修复效率下调影响基因组稳定性。此外，通过采用质粒重激活方法，我们发现SIRT6能够提高核苷酸切除修复（NER）效率，而其在黑色素瘤中的突变可导致细胞基因组来源于核苷酸切除修复缺陷的突变增多。后续机制研究发现SIRT6通过影响DDB2这一重要NER相关蛋白的去乙酰化提高NER修复效率。我们初步数据显示SIRT6可影响DDB2到达DNA损伤进而影响NER通路。基于该研究，我们也发现SIRT6高表达的宫颈癌患者在接受铂类药物治疗后预后更好，进一步提示SIRT6可通过NER效率防止铂类药物带来的负面影响。通过本项目的研究，我们发现SIRT6调控BER和NER通路调控衰老以及肿瘤发生和肿瘤进化的分子机制，为后续发展激活SIRT6的干预手段奠定了理论基础。