Rad6介导的修复途径参与端粒酶缺失酵母细胞中端粒重组的功能研究

Telomeres are special DNA-protein structures at the ends of eukaryotic linear chromosomes, which ensure genome integrity as they protect chromosome ends. In telomerase-null yeast cells, gradual telomere shortening and cellular senescence occur, while a very small portion of cells termed "survivors" can overcome the crisis via telomere maintenance by Rad52-dependent homologous recombination (HR). Although a dozen of proteins involved in DNA damage and HR-based DNA repair have been intensively investigated on recombination-dependent telomere elongation, little is known about effects of chromatin environment, including histone modifications and chromatin assembly, on telomeric recombination in the absent of telomerase. Here, we attempt to investigate involvement of Rad6-mediated repair pathways in telomere-telomere recombination (recombination efficiency and type) in telomerase-null yeast cells; reveal the molecular pathways and mechanisms involved in which Rad6–Bre1-mediated mono-ubiquitination participates in selection of survivor type (recombination type). This study focus on roles of Rad6-mediated repair pathways in telomere-telomere recombination, providing novel insight into the involvement of histone modification in telomere-telomere recombination and reference for understanding the mechanisms of recombination-mediated telomere elongation (alternative lengthening of telomeres, ALT) in cancer cells.

端粒是真核生物染色体末端的DNA-蛋白质复合体，对染色体末端的保护及确保基因组的稳定性具有重要作用。缺失端粒酶的酿酒酵母细胞可以通过依赖Rad52的同源重组方式维持端粒得以存活，即幸存子。目前，已经发现许多DNA损伤应激和同源重组相关的基因参与同源重组依赖的端粒复制，但对染色质环境（组蛋白修饰和染色质装配）是否参与端粒重组却知之甚少。本项目以酿酒酵母端粒为研究对象，结合遗传学、端粒PCR和端粒Southern杂交等手段系统研究Rad6及其下游三条途径参与端粒重组的功能；利用端粒Southern杂交、染色质免疫沉淀和端粒单链DNA检测研究Rad6–Bre1介导的组蛋白H2B的K123位点单泛素化调控端粒重组类型的分子机制。本研究将揭示Rad6介导的修复途径调控端粒重组效率和方式的分子机制，为理解组蛋白修饰参与端粒重组提供依据，并为认识依赖同源重组维持端粒的哺乳动物肿瘤细胞存活的机理提供借鉴。

端粒是真核生物染色体末端的DNA-蛋白质复合体，对染色体末端的保护及确保基因组的稳定性具有重要作用。缺失端粒酶的酿酒酵母细胞可以通过依赖Rad52的同源重组方式维持端粒得以存活，即幸存子。目前，已经发现许多DNA损伤应激和同源重组相关的基因参与同源重组依赖的端粒复制，但对染色质环境（组蛋白修饰和染色质装配）是否参与端粒重组却知之甚少。本项目执行过程中，我们紧紧围绕项目计划书预定目标，综合运用多种技术手段，对Rad6-Bre1介导的H2B单泛素化参与端粒复制、同源重组以及非带帽端粒保护的分子机制进行了系统研究，取得了多项重要研究成果：1）阐述了Rad6介导的三条修复途径端粒复制和同源重组的功能，并揭示了Rad6-Bre1-H2Bub1参与端粒复制和端粒同源重组的分子机制（Nucleic Acids Res 2017）；2）揭示了Rad6-Bre1-H2Bub1参与保护非带帽端粒的分子机制（DNA Repair 2018）；3）研究了亚端粒区域重要复合物Shelterin参与非带帽端粒保护功能的分子机制（J. Genet. Genomics 2018）。上述研究成果为理解组蛋白修饰参与端粒功能提供新的理论依据，并为认识哺乳动物ALT类癌症端粒重组提供有益的借鉴。