端粒解离酶ResT催化剪切莱姆病螺旋体端粒结构DNA分子机理的研究

Lyme disease pathogen Borrelia burgdorferi has a linear genome structure composed of over 20 replicons with covalently closed hairpin ends or telomeres. Telomere resolvase (ResT) plays as a permissive enzyme in replication intermediates of the linear genome. Telomere resolution is processed by 2-step transesterification reaction using a catalytic mechanism similar to that for type IB topoisomerases and tyrosine recombinases. Because of lacking the structure of ResT and ResT-DNA complex, the mechanism of how ResT bind prehairpined DNA forming a covalent 3’-phosphotyrosyl-ResT-DNA intermediate and catalyzes the DNA cleavage is still not clear.. In this project, the crystal structure of ResT and ResT-DNA complex will be determined by crystal X-ray. Domain structure of ResT will be analyzed to understand how DNA binding motif in N-terminal domain binds prehairpined DNA before DNA cleavage by studying the interaction of inframe deletions with DNA according to the crystal structure of ResT. The importance of conserved and semi-conserved residues during the DNA cleavage will be studied by measuring the DNA cleavage abilities of mutants. Comparing the structure of ResT and ResT-DNA complex, the conformational changes of the DNA binding motif will be confirmed and residues interacting with DNA will be studied by checking the DNA binding abilities of mutants.. Overall, the results from this project will not only promote further understand the mechanism of ResT binding, catalyzing and cleaving linear genome with covalently closed hairpin ends or telomeres, but also promote the development of ResT inhibitors.

端粒解离酶ResT在莱姆病螺旋体的端粒结构线性基因组DNA复制过程中起关键作用，是治疗莱姆病的靶蛋白。目前，由于缺乏ResT蛋白及ResT-DNA复合物的分子结构信息，ResT蛋白催化剪切端粒结构DNA的分子机理仍不清楚，这严重的限制了ResT蛋白小分子抑制剂的研发。本项目通过探索ResT蛋白及ResT-DNA复合物晶体的生长条件，获得高分辨率的蛋白晶体；利用X射线晶体衍射技术，解析ResT蛋白及ResT-DNA复合物的晶体结构，明确参与ResT蛋白特异性结合并剪切端粒结构DNA的关键氨基酸残基；利用荧光偏振和EMSA技术，研究关键氨基酸残基结合和剪切端粒结构DNA的特性，阐明ResT蛋白催化剪切端粒结构DNA的分子机理。本研究为基于ResT-DNA复合物晶体结构的计算机辅助药物设计，筛选和优化ResT蛋白小分子抑制剂治疗莱姆病提供科学依据。

莱姆病（Lyme disease）是人畜感染莱姆病螺旋体（Borrelia burgdorferi）引起的传染性疾病，每年都有万余病例报道，给人类的生产和健康带来巨大威胁。端粒解离酶ResT（telomere resolvase，ResT）在莱姆病螺旋体端粒结构线性基因组DNA复制中起关键作用，是治疗莱姆病的靶蛋白。由于缺乏ResT-DNA复合物的晶体结构信息，且ResT剪切莱姆病螺旋体基因组DNA的分子机理仍不清楚，因此限制了ResT小分子抑制剂的研发。本项目以ResT为研究对象，通过分析不同表达菌株中ResT可溶性蛋白的表达量筛选出最优的原核表达菌株；利用定点突变获得ResT（3-449） W94H/Y210S突变体，并纯化该突变体获得高纯度且状态均一的ResT蛋白；利用EMSA检测了ResT 蛋白与DNA结合的能力；使用蛋白晶体接种（seeding）和交叉接种（cross-seeding）的方法获得ResT蛋白晶体；利用X-射线衍射分析解析了ResT（3-449） W94H/Y210S晶体结构，为研究ResT剪切基因组DNA分子机理奠定基础。同时，申请人研究了苯乙烯单加氧酶合成对映纯环氧化物的催化机制；首次解析了来源于黄曲霉的嗜热烯还原酶AfOYE1及间甲基转移酶AfvC的晶体结构，为实现AfOYE1烯烃不对成还原工业化应用奠定基础，也为研究黄曲霉依赖间甲基转移酶AfvC合成次级代谢产物Aflavarin的合成机理提供理论依据。