【作者】 华孝挺；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2010， 博士

【摘要】 耐辐射球菌能在电离紫外辐射、化学毒剂、热、干燥等极端环境下存活。这得益于它不同寻常的DNA修复能力,特别是对电离辐射导致的双链断裂的重组修复。这种难以置信的DNA修复能力令D. radiodurans成为很好的模式生物。尽管如此,该细菌的修复机理至今仍知之甚少,需要进一步研究。RecQ家族的DNA解旋酶对基因组稳定性的维持起到必要的作用。耐辐射球菌中的RecQ是RecQ家族中特别的一员,不同于其他物种来源RecQ的是,它在C端有着三个串联HRDC结构域。而与RecQ蛋白互作的单链DNA结合蛋白(SSB)在所有的DNA代谢进程中非常重要,包括DNA复制,重组和修复。与其他细菌SSB蛋白均以同源四聚体行使功能不同,耐辐射球菌SSB是同源二聚体的,每个单体含有两个OB fold.本文主要系统研究耐辐射球菌RecQ和SSB蛋白的生化活性及突变分析系统分析。具体结果如下：1.首先构建了RecQ突变株。RecQ突变株对辐照、UV、过氧化氢和丝裂霉素C均非常敏感。通过补偿突变株以RecQ的不同部分,发现解旋酶和三个HRDC结构域对DNA损伤抗性均为不可缺少。解旋酶结构域对解旋酶活性和ATP酶活性均为必需,而三个串联HRDC结构域则促进了这些活性。2.基于其晶体结构构建四个SSB截断突变体。凝胶过滤结果表明DrSSB、DSCT和DSCC是同源二聚体,而DSN和DSNC是单体。这支持了N端在二聚体形成过程中起主要作用的假设。我们使用EMSA和FRET对每个OBfold在DNA结合中的作用进行了探索。3.在uvsE uvrAl uvrA2的基础上,敲除了recQ构建了四突变株。用rpoB/Rif系统测量了野生株、突变株recQ(MQ)、uvsE uvrA1 uvrA2 (TNK006),和uvsE uvrA1 uvrA2 recQ(TQ)的突变频率和突变率。然后分离Rif的突变体,并对rpoB基因进行了测序。结果表明,recQ通过与uvsE uvrA1 uvrA2的互作参与了紫外损伤修复途径。4.测定在不同利福平浓度下(从5到50μg/ml的浓度梯度)的突变率。耐辐射球菌在5μg/ml利福平筛选压力下的突变率显著高于其在25μg/ml和50μg/ml下的突变率。耐辐射球菌突变的利福平浓度依赖效应,不仅表现在突变率上,也表现在突变谱上。我们推测,在低利福平浓度下,耐辐射球菌重点防止碱基替换突变,因为活性氧ROS主要造成氧化碱基损伤。5.使用DNA芯片技术和生化实验来鉴定耐辐射球菌p亚基中突变是如何通过调控其他基因转录来改变生长速度的。可从这些结果中分析p亚基中突变是如何调控其他基因转录的分子机制。综上所述,RecQ解旋酶的三个HRDC结构域不仅对DNA损伤抗性不可缺少,对解旋酶和ATPase活性也很重要。其互作蛋白SSB的ssDNA结合活性需要两个OB fold的互作。突变分析系统的研究表明了RecQ与紫外损伤修复系统的互作关系。耐辐射球菌突变的利福平浓度依赖效应,不仅表现在突变率上,也表现在突变谱上。芯片分析则展示了β亚基中突变是如何调控其他基因转录。更多还原

【Abstract】 Deinococcus raidoudrans can survive extreme DNA damage caused by ionizing radiation and other DNA damage agents at levels that would otherwise kill Escherichia coli and other related organisms. The survive of D. raidoudrans in such extreme conditions is due to its extraordinary DNA repair ability, especially in the reconstruction of dsDNA breaks induced by ionizing radiation. The incredible DNA repair capacity makes D. radiodurans an excellent model system. However, current studies have only touched the surface, and much is still to be learned from D. radiodurans.The RecQ family of DNA helicases performs essential functions in the maintenance of genomic stability in all organisms. In D. radiodurans, DR1289 is a special member of RecQ family with unique arrangement of three tandem HRDC domains in the C-terminus. Moreover, single stranded binding protein (SSB) that interacted with RecQ plays crucial in all kind of DNA metabolic processes includeing DNA replication, recombination and repair. In contrast to other bacterial SSB proteins, which are functionally active in homotetramers, Deinococcus SSB-like proteins are homodimers in nature and contain two OB fold per monomer. In this study, we investigated biochemical characterization and mutational pattern of recQ and its related gene in D. radiodurans. The results were listed as below:1. RecQ mutant was constructed at first. A dr1289 mutant is hypersensitive toγ-irradiation, UV, H2O2 and mitomycin C. 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 complete DNA damage resistance. We also found that the helicase domain is necessary for the unwinding and ATPase activity, and the three tandem HRDC domains increase the efficiency of these activities.2. Four truncated variants of DrSSB proteins were generated based on their crystal structures. Gel filtration showed that DrSSB, DSCT and DSCC were homodimers, and DSN and DSNC were monomers. The gel filtration analysis supported the hypothesis that the N-terminal domain played a predominant role in dimerisation. Biochemical characterization was used to determine the role of each OB fold in DNA binding, by electrophoretic mobility shift assay (EMSA) and fluorescence resonance energy transfer analysis (FRET).4. A four-gene mutant strain was created by constructing recQ knockout mutant in uvrAl, uvrA2, and uvsE knockout backgrounds. Using the rpoB/Rif system, we measured the mutation frequencies and rates in wild type, recQ (MQ), uvsE uvrAl uvrA2 (TNK006), and uvsE uvrAl uvrA2 recQ (TQ). We then isolated Rif mutants of these strains and sequenced the rpoB gene. The results indicate that recQ is involved in the ultraviolet damage repair pathway via the interaction between recQ and uvrAl, uvrA2, and uvsE in D. radiodurans.5. The mutation frequencies and rates of D. radiodurans were measured at a wide concentration range from 5 to 50μg/ml of rifampin. We found that the mutation rate of the bacterium in the presence of 5μg/ml of rifampin was significantly higher than in the presence of 25μg/ml and 50μg/ml rifampin. Rifampin had concentration-denpendent effect not only on the mutation rate, but also on the mutation spectrum. It is speculated that D. radiodurans mainly prevents base substitution mutation at low concentration of rifampin, since ROS induced mainly oxidative base damage6. DNA microarrays and biochemical assays were used to identify the molecular details of how the Rif mutation in the P-subunit led to changes in growth rate via altered regulation of multiple genes in D. radiodurans. The results provided a molecular insight into the mechanism of Rif mutation in theβ-subunit on altering the regulation of multiple genes.Taken together, the three HRDC domains of RecQ helicase is important not only for radiation resistance, but also for helicase activity and ATPase activity of RecQ. SSB that interacted with RecQ needed the interaction between two OB folds. Mutational analysis suggested that recQ is involved in the ultraviolet damage repair pathway. Rifampin had concentration-denpendent effect not only on the mutation rate, but also on the mutation spectrum in D. radiodurans. The microarray results provided a insight into the mechanism that Rif mutation inβsubunit alter the regulation of multiple genes.更多还原