【作者】 郑嘉熙；

【导师】 汪世华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 XPB/Ercc3是真核生物中TFIIH复合体的一个亚基,编码一个3’→5’方向的解螺旋酶,具有ATP依赖性的3’→5’helicase活性和ssDNA依赖性的ATPase活性。XPB作为TFIIH的一个组分参与转录起始与核苷酸切除修复（NER）,分别负责转录泡的打开与包含损伤位点的内切片段的移除。XPB作为细胞内关键途径的感受器和效应器还参与了细胞周期的调控和其他调控途径。在分枝杆菌属的原核生物中,通过同源比对发现了ercc3基因的同源编码序列,但未发现在真核中与ercc3相互作用并共同行使功能的那些基因的同源序列。并且,分枝杆菌有其自有的转录起始机器和核苷酸切除修复系统。因此,ercc3在分枝杆菌中的功能就成为一个需要阐明的问题。本文利用基于噬菌体基因的分枝杆菌重组工程系统,将串联亲和标签protein A-TEV protease site-calmodulin binding protein（TAP tag）定点敲入至耻垢分枝杆菌的ercc3基因表达框C末端,使带有TAP标签的Ercc3蛋白在基因组天然启动子的调控下表达,其表达量,定位与调控水平都和野生型ercc3基因一样。然后我们利用标签与两种不同亲和配体的结合作用,经过两次亲和纯化分离细胞裂解液中的Ercc3蛋白。由于整个纯化过程在温和条件下进行,与Ercc3蛋白相互作用的蛋白也被一起纯化出来。纯化组分经过跑SDS-PAGE分离,银染染色后,切下特异性的条带,trypsin酶解后打质谱分析。从质谱结果中我们得到了一系列蛋白,我们选择了其中的DNA依赖性RNA聚合酶β’亚基RpoC和β亚基RpoB,以及同源重组蛋白RecA进一步研究。经过farwestern和SBP pull-down验证,我们发现Ercc3与RNA聚合酶β’亚基RpoC在体外有直接的相互作用。在与实验室其他成员的合作中发现,Ercc3蛋白还与预测为DNA repair related ATPase的ATP依赖性的DNA结合蛋白ATPase,和预测为转录因子的共操纵子邻近基因MSMEG₅705有相互作用。但ercc3基因的敲除尚未发现明显的表型。根据以上结果,我们推测, ercc3基因可能与其相互作用蛋白共同参与耻构分枝杆菌的转录起始,并且很可能是介导耻垢分枝杆菌,以及结核分枝杆菌在特殊生存状态下的转录调控。更多还原

【Abstract】 Eukaryotic XPB/Ercc3 is a subunit of protein complex TFIIH which participate in transcription initiate, promotor clearance and nucleotide excision repair（NER） of DNA damage. XPB uses its ATP-dependent helicase activity and ssDNA-dependent ATPase activity to play a role in the opening of DNA double strand in transcription and remove of damage contained DNA fragment in NER. XPB also participate cell cycle regulation and other regulation pathway as a molecular sensor and responsor to a lot of cell pathways. In prokaryotic life Mycobacterium, we found the homologue of eukaryotic ercc3, but no other transcription and NER relative gene which interact and co-work with eukaryotic ercc3. Mycobacterium has its prokaryote transcription and NER system of itself. So the function of ercc3 gene in Mycobacterium is still to a question at issue.By a Mycobacterim recombineer system base on phage gene, we inserted TAP tag （protein A-TEV protease site-calmodulin binding protein） into C teminal of ercc3 gene in M.smegmatis genome. As a result, the recombinant M.smegmatis express ercc3-tap gene as the wild type ercc3 gene, since they are both simple copy and transcripted by the same promotor. Used the method of TAP（tandom affinity purification）, we purified the Ercc3-TAP and its interaction protein from cell extraction. After SDS-PAGE and silver stain, the purification of interaction protein was identified by LC-MS.The LC-MS result revealed a list of possible interaction protein with Ercc3, include DNA-directed RNA polymeraseβ’andβsubunit （rpoC and rpoB）, and recombinant protein recA. By in-vitro test such as farwestern and pull-down, we found Ercc3 interaction directly with RpoC in-vitro. And other experience found Ercc3 also interact with hypothetical protein ATPase and MSMEG₅705. But we didn’t find distinct phenotype on ercc3-delected strain. From the data above, we can deduce that ercc3 （with its interaction partner） may participate in transcription initiation, particularly transcription in some special environment.更多还原