【作者】 黄丽芬；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2006， 博士

【摘要】 耐辐射球菌（Deinococcus radiodurans）是迄今为止发现的对电离辐射、紫外线、干燥、过氧化氢等一些DNA损伤剂都具有极强抗性的微小球菌，它在DNA双链断裂修复上具有超强的能力，是目前研究DNA损伤与修复的模式生物。从1999年Deinococcus radiodurans野生株R1全基因组数据公布以来，该细菌的分子结构特征研究，分子防御机制方面和重要修复基因，如recA、ssb、pprI、pprA等在DNA修复机制方面都取得了很大的进展，但还未能清楚揭示耐辐射球菌的修复机理。RecQ解螺旋酶是解螺旋酶在进化中高度保守的一个亚族，它在维持有机体基因组的稳定性中有重要的作用。耐辐射球菌具有两个特殊结构的RecQ家族成员：DR1289和DR2444，这两个基因在该细菌中的具体功能都未知。本文主要系统地研究这两个基因在耐辐射球菌中的作用机制和调控网络，具体从以下几个方面进行了研究：1．先运用生物信息学的方法确定这两个基因在染色体上的具体位置，对DR1289和DR2444进行了结构域的同源性分析，功能预测和进化树分析，结果表明DR1289与细菌及真核生物有较近的亲缘关系，而DR2444相差较远。2．运用PCR突变三段连接法克隆具有自身groEL启动子、KAT启动子分别与卡那霉素抗性基因、氯霉素抗性基因融合的DNA片段反向重组到基因组中，构建并鉴定了卡那霉素抗性完全突变株ΔDR1289，氯霉素抗性完全突变株ΔDR2444，双突变株ΔrecQ。辐射条件下和H₂O₂氧化压力下突变株生存率结果表明：ΔDR2444与R1存活率趋势线基本一致，而ΔDR1289和ΔrecQ双突变株较为敏感。根据上述结果推测，DR1289是一个对R1保持极端抗性的必需基因，而DR2444是非必需基因。3．分析了突变株ΔDR1289的一些特性，表型数据表明，敲除掉DR1289这个基因后，突变株对UV，丝裂霉素C，过氧化氢都极敏感，对γ-辐射在超过2 kGy剂量下敏感。ΔDR1289生长与野生株相比较为缓慢；脉冲场电泳表明，无论在正常生长状况，还是过氧化氢胁迫条件下，该突变株都具有基因组的不稳定性，尤其是在20mM H₂O₂处理30分钟后，其基因组图谱与R1在高剂量辐射和长期干燥胁迫下的图谱很相似。运用穿梭质粒pRADK进行DR1289各个结构域的功能性补偿，表明helicase结构域和3个串联的HRDC结构域都必不可少。4．用大肠杆菌表达系统体外表达了DR1289各个结构域的突变蛋白，并进行了纯化。我们使用荧光染料H33258，长度为2.7 kbp的线性化双链（HindⅢ-pUC19）片断作为底物的荧光染色替代法检测解螺旋酶活性。不同ATP和金属离子浓度下该蛋白的解螺旋酶活性表明，DR1289的解螺旋酶活性至少与曾经报道过的大肠杆菌的RecQ活性相当。SSB蛋白作为一个解螺旋酶功能促进因子，我们发现DR1289解螺旋速率能够被D．radiodurans SSB蛋白提高，且对DR1289蛋白有相同物种专一性的特殊效应。ATPase活性表明helicase结构域对DR1289 ATPase是必需的，而HRDC结构域是达到最适ATPase酶活性所必需的结构域。基于这些实验，我们推测在耐辐射球菌中，DR1289的C-末端三个串联的HRDC结构域已经进化，从而应对各种类型的DNA损伤。5．在DR1289删除以后，全基因组转录谱水平约有9.1％发生了2倍以上的改变，如铁离子的代谢，氧化相关基因和细胞分裂相关基因。另外，在20mM H₂O₂压力下，突变株许多重要基因如recA，pprA，ddrA，DR0003，DR0070的转录水平变化趋势与野生株在强剂量辐射和长期干燥条件下的趋势很相象。Western blot分析表明无论在有氧化胁迫条件，还是在正常生长条件下，突变株中RecA水平都上升，表明当DR1289删除后，体内确实存在较大的DNA损伤，进一步佐证了芯片数据。高锰低铁可以促使耐辐射球菌的抗性提高，但ICP-MS测定结果显示DR1289突变体内全铁含量上升，而全锰含量下降，导致Mn／Fe比例从0.38下降到0.07。而顺磁共振实验表明突变株中的自由铁量约为野生株的50％。综上所述，DR1289基因是一个维持耐辐射球菌基因组稳定性和贡献于极端抗性的关键基因。6．为了进一步研究耐辐射球菌DR1289蛋白在极端抗性中的调控机制，通过二维液相色谱ProteomeLab PF 2D目标蛋白快速分离系统分析了野生株R1与功能破坏株ΔDR1289在20 mM H₂O₂氧化压力下蛋白表达谱的差异。综上所述，耐辐射球菌的两个recQ基因中，DR2444是不参与极端抗性的非必需基因，而DR1289行使着多项重要的功能，体内活性、体外活性、转录谱表达和蛋白谱表达实验均表明它能够维持耐辐射球菌基因组的稳定性，保持细胞体内稳态性且和多个重要基因存在互作，是贡献于耐辐射球菌超强修复能力的关键基因。更多还原

【Abstract】 The bacterium Deinococcus radiodurans is extremely resistant to ionizing radiation, ultraviolet, hydrogen peroxide and many other agents that damage DNA. Studies show that this resistance is due to D. radiodurans’s extremely proficient and accurate DNA repair process. After the release of genome sequence of D. radiodurans wild type strain R1 in 1999, the structure characteristics, molecular defense systems and the biochemical mechanisms of many key repair genes, such as recA、ssb、pprI、pprA are making great progress. But the accurate regulatory network of DNA repair in Deinococcus radiodurans is still remaining unknown in large part.As a member of SF1 superfamily, the RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. D. radiodurans encodes two recQ genes with unusual domain, DR1289 and DR2444, whose functions, however, remain obscure currently. In this paper, the biochemical mechanism and regulatory network of the two genes were studied thoroughly. The main work and its results are appended following:1. Using bioinformatic methods, the loci of DR1289 and DR2444 in the chromosome were localized. Sequence alignment of different domains, biochemical function prediction and construction of phylogenetic tree were performed. Results obtained from all above studies suggest that DR1289 has a close phylogenetic relationship with conserved RecQ family members from bacteria to eukaryotic organisms, whereas DR2444 has distant relationship with RecQ family.2. A fusion DNA fragment carrying kanamycin resistance gene with the D. radiodurans groEL promoter, chloramphenicol resistance gene with KAT promoter was cloned by PCR amplification and inserted into the recQ locus in the genome of the wild-type strain R1. Three resulting recg-deficient strains, designated △DR1289,△DR2444 and△recQ （double mutation）, were constructed. Results show that△DR1289 and△recQ were very sensitive to ionizing radiation and H₂O₂, while△DR2444 was not. The phenotype of△DR1289 was similar to many RecQ helicase mutants. Therefore, it was presumed that DR1289 was the necessary gene in maintaining the extreme resistance to DNA damaging agents, whereas DR2444 was not. Further research based on genetic and biochemical approaches should help togain a better understanding of the genes involved in DNA repair.3. Some phenotypes of mutant△DR1289 were characterized. The mutant strain△DR1289 was sensitive to mitomycine C, hydrogen peroxide and UV. Interestingly, when the dosage of gamma radiation up to 2 kilograys, the radiation resistance of the mutant decreased remarkably compared to that of the wild type R1. After the deletion of DR1289, growth rate of the mutant was significantly slower. In addition, results of pulsed-field gel electrophoresis （PFGE） showed that the genome stability was destroyed in DR1289 mutant. After 20 mM H₂O₂ treatment for 30 minutes, genome mapping of the mutant presented in PFGE was very similar to R1 treated with high dose gamma irradiation and prolonged desiccation. By complementing the DR1289 mutant with various domains of DR1289 in vivo, we have determined that the helicase and all three HRDC domains are indispensable for DNA damage resistance.4. The DR1289 protein and its variant proteins were expressed by E. coli expression system and then purified. Using a continuous fluorescent dye-displacement assay, we investigated the optimal conditions for DR1289 unwinding function at various concentrations of ATP and metal ions, indicating that the helicase activity is comparable to that observed of E. coli RecQ. SSB protein servers as a stimulatory factor of the unwinding by DR1289. The unwinding rate of DR1289 increases by addition of D. radiodurans SSB protein, DrSSB has a species-specific effect on DR1289. We also found that the helicase domain is necessary for the ATPase activity and that the three tandem HRDC domains increase the efficiency of these activities. Based on these data, we propose that the C-teiminus of DR1289 has evolved in D. radiodurans to confront the types and amounts of DNA damage caused by this organism’s extreme environment.5. Global transcriptome profiles response to DR1289 deletion showed that 9.1% of genome changed at least 2-fold in DR1289 mutant strain, such as iron homeostasis, oxidative related genes, and cell division related genes. Furthermore, under 20mM H₂O₂ stress, the profile expression patterns of many genes （e.g., recA, pprA, ddrA, DR0003, DR0070） were similar to acute gamma-irradiation and prolonged desiccation. Western blotting results also demonstrated that RecA was up-regulated in DR1289 mutant, indicating that there were vast DNA damages in vivo. Mn （II） accumulation （with low Fe） facilitates resistance in D. radiodurans, but in DR1289 mutant, the intracellular level of Fe is higher, Mn is lower, which decreased the Mn/Fe ratio significantly, whereas free iron in DR1289 mutant in only fifty percent of wild type by EPR experiment. Taken together, DR1289 is a key gene in maintaining the genome stability and contributing the extraordinary ability to D. radiodurans.6. To further unlocking the regulatory mechanism of DR1289 in D. radiodurans, ProteomeLab PF 2D protein quick separation method has been developed for differential display of proteins from cell lysates and applied to a comparison of protein expression between wild type and DR1289 mutant in 20 mM H₂O₂ stress.Taken together, among the two recQ genes in D. radiodurans, DR2444 is not the necessary gene to extreme resistance, on the contrary, DR1289 acts as a key gene with multiple roles in vivo and in vitro, which is required for maintaining genome stability, cellular homeostasis and interaction with many important genes.更多还原