【作者】 周庆；

【导师】 华跃进； 徐步进； 唐孝威；

【作者基本信息】 浙江大学 ， 生物物理学， 2007， 博士

【摘要】 耐辐射球菌不仅对电离辐射具有极端的抗性，而且它对紫外线、过氧化氢、干燥以及其它物理化学诱变和胁迫因子也具有很高的抗性。迄今为止，我们对于它的极端抗性的分子机制了解得还很有限。对耐辐射球菌中与极端辐射抗性相关的蛋白的研究，将有助于我们加深对耐辐射球菌这一极端抗性的理解。本论文从4个方面对耐辐射球菌进行了较为系统的研究，获得的结果如下：(1)耐辐射球菌RadA参与了DNA损伤修复过程RadA是一个高度保守的蛋白，属于RecA／RadA／Rad51蛋白超家族。这个蛋白超家族在细菌，古细菌和真核细胞中均被发现。在古细菌中，RadA蛋白具有RecA类似的功能并在同源重组过程中起关键作用。在大肠杆菌中，RadA蛋白也参与了重组和DNA损伤修复过程，但远没有在古细菌中的功能那么重要。用PSI-BLAST方法，我们发现，与古细菌以及真核细胞中RadA蛋白相比，耐辐射球菌RadA蛋白和细菌中的RadA的相似性更高。研究发现耐辐射球菌radA基因的突变增加了耐辐射球菌对γ射线和紫外线照射的敏感性，但对过氧化氢氧化胁迫的抗性没有影响。耐辐射球菌和大肠杆菌radA基因均能完全补偿突变体对γ射线和紫外线辐照的抗性。进一步的结构域功能分析表明，和radA突变体的抗性相比，锌指结构域的缺失导致耐辐射球菌对γ射线和紫外线辐照更加敏感；仅仅缺失Lon蛋白酶结构域的耐辐射球菌表现为比radA突变体略微更抗γ射线和紫外线辐照处理。这些实验结果表明，耐辐射球菌RadA蛋白的确参与了DNA损伤修复过程，而且不同的结构域具有不同的功能。(2)耐辐射球菌RecD参与了抗氧化过程RecBCD在许多生物体的双链DNA损伤修复过程中起关键作用。在耐辐射球菌中，这个蛋白酶复合体是不完整的。它的基因组只编码一个RecD蛋白，而缺少了RecB和RecC。对耐辐射球菌RecD的生化分析表明：它具有5’-3’方向的解螺旋酶活性，但是这种活性很低。在本研究中，我们构建了耐辐射球菌recD的突变体并研究了它可能的生物学功能。研究结果显示：与野生型R1相比，recD基因的突变使得耐辐射球菌对过氧化氢氧化胁迫处理极其敏感而且略微降低了耐辐射球菌对紫外线辐照的抗性，然而对耐辐射球菌的电离辐射抗性没有任何影响。补偿实验结果显示，只有完整的耐辐射球菌RecD或者耐辐射球菌RecD的解螺旋酶结构域和N-末端结构域共同过量表达才能够完全补偿recD基因的突变菌株对过氧化氢氧化胁迫的抗性，而大肠杆菌RecD、单独的耐辐射球菌RecD的解螺旋酶结构域或耐辐射球菌RecD的N-末端结构域都不能补偿这一表型。进一步的实验结果表明：RecD通过调节过氧化氢酶活性和活性氧自由基清除活性参与了耐辐射球菌的抗氧化过程。(3)耐辐射球菌不具有经典的SOS反应LexA和RecA在大肠杆菌经典的SOS反应过程中起关键作用。LexA作为一个转录抑制因子控制着包括RecA在内的许多基因的DNA损伤后的诱导表达。以前的研究表明，耐辐射球菌中单独LexA1和LexA2都不参与RecA的诱导表达。在本研究中，我们构建了耐辐射球菌lexA1和lexA2基因的双突变。研究结果显示，与野生型和两个单突变体相比较，lexA1和lexA2基因双突变明显降低了耐辐射球菌的生长速度，而且增加了耐辐射球菌对紫外线辐照和过氧化氢氧化胁迫的抗性。在正常生长情况下，双突变并没有使耐辐射球菌细胞内RecA本底表达量明显上升，而是和野生型和两个单突变体中的RecA蛋白水平一致。这表明LexA1和LexA2一起也不参与对RecA蛋白诱导表达的调控。进一步的DNA芯片数据显示，LexA1和LexA2一起参与了包括细胞分裂、蛋白合成、抗氧化和转录调控等许多分子机制的调控过程。以上实验结果表明，耐辐射球菌并不具有经典的SOS反应，它应该利用一种新的分子机制来调控DNA损伤诱导蛋白的诱导表达过程。(4)耐辐射球菌PprI通过拮抗RecX的功能来调控RecA和PprA的诱导表达RecA在同源重组过程中起重要作用，以便在DNA受到严重损伤时及时修复那些DNA双链断裂；PprA则在非同源末端连接过程中起作用。到目前为止，有关耐辐射球菌RecA和PprA的诱导表达分子机制还不清楚。以前的研究显示：在RecA的转录调控过程中，PprI作为一个正调控因子起作用，而RecX则作为一个负调控因子起作用。在本研究中，我们构建了pprI／recX双突变体，并揭示了它们之间的调控关系。我们发现pprI／recX双突变可以完全恢复pprI突变体对紫外线和过氧化氢处理的抗性，但不能恢复pprI突变体对γ射线辐照处理的抗性。进一步的补偿实验结果表明：RecA的过表达也能完全补偿pprI突变体对紫外线和过氧化氢处理的抗性，而PprA的过表达不能补偿pprI突变体的三种抗性中的任何一种抗性。另外，重组频率和免疫印记分析结果表明：耐辐射球菌recX的突变可以上调RecA和PprA在正常生长条件下的本底表达水平。以上结果说明，耐辐射球菌PprI通过拮抗RecX的负调控作用来调控RecA和PprA的诱导表达。更多还原

【Abstract】 The extremely radio-resistant bacterium, Deinococcus radiodurans, is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet, hydrogen peroxide, desiccation and other physical and chemical DNA-damaging agents. However, little is known about the molecular mechanisms responsible for the extraordinary resistance in this organism. In this study, the functions and regulation of these proteins involved in DNA damage repair were investigated. The results as below:1 RadA takes part in the DNA damage repair process in D. radioduransRadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein superfamily found in bacteria, archaea and eukaryota. In Archaea, it plays a critical role in homologous recombination process due to its RecA-like function. In Escherichia coli, it takes part in conjugational recombination and DNA repair but is not as important as that in archaea. Using PSI-BLAST searches, we found that D. radiodurans RadA has a higher similarity to that in bacteria than that in archaea and eukaryota. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet, but had no effect on the resistance to hydrogen peroxide. Complementation of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant, while the absence of the Lon protease domain exhibited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.2 D. radiodurans RecD has a new role in antioxidant processRecBCD holoenyme plays a critical role in double-stranded DNA breaks in many organisms. In D. radiodurans, this complex is not intact because of the absence of RecB and RecC, but a RecD-like protein does indeed exist. Biochemical analysis has been shown that it is a 5’-3’ helicase and has a low processivity. In this work, we constructed D. radiodurans recD disruptants and investigated their possible biological functions. The results showed that disruption of the recD gene of D. radiodurans resulted in remarkably increased sensitivity to hydrogen peroxide and slightly decreased resistance to ultraviolet radiation, but had no apparent effect on the resistance to gamma radiation compared to the wild type. Furthermore, complementation experiments showed that E. coli RecD, helicase domain or N-terminal domain of D. radiodurans RecD couldn’t individually restore the resistant phenotype to hydrogen peroxide of the D. radiodurans recD disruptant, whereas the complete D. radiodurans RecD protein and co-expression of the helicase domain and the N-terminal domain of D. radiodurans RecD could, indicating that the integrity of D. radiodurans RecD is required for this function. Further studies showed that D. radiodurans RecD took part in antioxidant process by stimulating catalase activity and reactive oxygen species scavenging activity in D. radiodurans. These results suggest that D. radiodurans RecD has a new role in the antioxidant pathway.3 D. radiodurans doesn’t have a classical SOS responseLexA and RecA play a crucial role in SOS response in E. coli. In classical SOS response, LexA functions as a repressor to regulate the expression of many genes including RecA after DNA damage occurs. Previous studies showed that both LexAl and LexA2 are not involved in RecA induction in D. radiodurans. In this study, we constructed a double mutant of lexAl and lexA2. Results showed that mutation of lexAl and lexA2 resulted in an apparent decrease in growth rate and an increased resistance to ultraviolet and hydrogen peroxide compared with wild type Rl and two single mutants. However, this double mutant did not exhibit any remarkably increased constitutive expression of RecA under normal conditions, just like wild type Rl and two single mutants, suggesting that neither LexAl nor LexA2 is involved in the induced expression of RecA. Further transcriptional assay showed that LexAl and LexA2 together participated in many regulatory pathways involved in cell division, protein synthesis, antioxidant process, transcription regulation andother mechanisms. These results indicate that there should be a new mechanism bywhich these damage-induced proteins are regulated in D. radiodurans.4 PprI regulates the induced expression of RecA and PprA via antagonizingRecXRecA plays a central role in homologous recombination repair to mend double-strand DNA breaks when DNA is highly damaged and PprA is involved in the non-homologous end-joining repair pathway. To date, little is known about the biochemical mechanism controlling the expression of D. radiodurans RecA and PprA. Our previous work showed that PprI is a positive factor and RecX is a negative factor in the transcriptional regulation of D. radiodurans RecA. Here, we found that the double mutation of D. radiodurans pprI and recX could restore the resistance of the pprl mutant to ultraviolet and hydrogen peroxide but not to gamma radiation. Furthermore, complementation of the pprI mutant by D. radiodurans RecA also could fully recover the resistance of the pprI mutant to ultraviolet and hydrogen peroxide, however, D. radiodurans PprA couldn’t compensate all three kinds of resistances of the pprI mutant. In addition, recombination rate analysis and Western blotting results showed that mutation of D. radiodurans recX could up-regulate the basal expression levels of D. radiodurans RecA and PprA under normal conditions. Taken together, these results suggest that PprI regulates the induced expression of RecA and PprA via antagonizing RecX function in this bacterium.更多还原