【作者】 郭晓飞； 李辉； 曹恩华；

【Author】 GUO Xiaofei, LI Hui, QIN Jingfen, CAO Enhua * (Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China)

【机构】 中国科学院生物物理研究所；

【摘要】 人类端粒DNA是由5–15kb的串联的重复序列(TTAGGG)n组成,3’末端为单链悬突(3’-overhanging)并回折形成环形(T-loop)结构,保护3’单链悬突,防止染色体末端降解、重组和融合。最近发现DNA损伤后端粒结合蛋白TRF2位于双链断裂处,可能参于DNA修复。到目前为止,还没有证据表明TRF2蛋白参于损伤的端粒T-loop重建。本工作中,我们制备了四种(3’-单链悬突、平端、O6MeG-和8-HOG-)含(TTAGGG)96的线性端粒DNA模型。运用原子力显微镜(AFM)技术在纳米尺度上直接观察TRF2蛋白与上述模型端粒DNA的相互作用。结果表明TRF2蛋白能与3’-单链悬突端粒DNA结合并形成三种不同类型T-loop结构。I型结构类似于细胞内端粒T-loop结构,其环-尾结合处DNA为三链结构,夹角通常大于90度。而II型和III型的两个线性双链以纽结方式形成T-loop,其环-尾结合处DNA多为四链结构,夹角通常小于90度。首次观察到TRF2蛋白也能够使损伤的线性平端-、O6MeG-和8-HOG-端粒DNA形成不同类型T-loop结构。此过程不依赖于3’单链悬突的存在,也不要求额外的能源。我们的发现提出了一种可能性,在细胞内TRF2蛋白可通过一种错配方式使损伤DNA重新折叠形成一种不稳定无功能的T-loop。提示细胞内异常T-loop的形成可能是端粒DNA损伤后TRF2蛋白错误修复的结果。这种新的作用不同与先前提出的TRF2蛋白对端粒的保护作用。更多还原

【Abstract】 Human telomeres typically consist of 5–15 kb of (TTAGGG)n tandem repeats and terminate in a 3’ single strand tail. This tail is proposed to loop back and invade the telomeric duplex tract resulting in a T-loop which protects the single-stranded 3’ chromosome ends from recombination, fusion, and from being recognized as damaged DNA. Recently, it reports TRF2 might localize to double-strand breaks after DNA damage and involved in various DNA repair pathways. However, it is not any evidence as yet if the damaged telomeric DNA could be remodeled into a loop by TRF2 protein. Here, we prepared four model DNA carrying a stretch of telomeric sequence (TTAGGG)96 with 3’ -overhanging, blunt ended, O6MeG- and 8-HOG- substitution, and then observed their binding capacity of TRF2 protein and T-loop formation by atomic force microscopy. Results elucidated that the telomeric DNA with 3’ –overhanging could form three type of T-loops by TRF2 in vitro. Type I was similar to a typical T-loop structure in cell, with a tri-stranded junction and an angle of larger than 90 degrees between the two loop-forming DNA strands. Type II and III showed a t-loop/ braided knot structure with a tetra-stranded junction and an angle of less than 90 degrees. Further studies found in vitro TRF2 also could remodel the blunt ended, O6MeG- and 8-HOG-substituted telomeric DNA into various types of loops by above braided knot model. Its formation is independent of the end-structure, not require other energy sources. These results demonstrate that damaged telomeric DNA with TRF2 could form a unstable T-loop in vitro by an error-prone assembly, implicating the formation of abnormal telomeric T-loop in cells probably is a result of an error-prone assembly. Our results revealed a possible role of TRF2 that are different from protection of T-loop previously.更多还原