酵母转录因子Cdc73的结构生物学研究
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摘要
Pafl复合物(PaflC)是一种在真核生物中非常保守的复合物,目前已获得鉴定的PaflC中,含有Paf1、Cdc73、Ctr9, Leol及Rtfl(最近发现hSki8也是其人源复合物的组分之一)五种蛋白质组分。 PaflC主要的功能是通过与RNA聚合酶Ⅱ (RNApolⅡ)相互作用,共同参与包括转录起始、延伸和终止的全过程。作为Pafl复合物中最早被鉴定出的亚基之一,酿酒酵母Cdc73蛋白参与了很多转录相关的生命过程,包括与RNA Pol Ⅱ的直接相互作用,招募和激活组蛋白修饰相关因子并和其它的一些转录激活因子共同参与转录。酿酒酵母Cdc73蛋白在人源中的同源蛋白Parafibromin被鉴定为一个肿瘤抑制因子,它的突变与乳腺癌,肾癌和胃癌的发生相关。
     本文第一部分以酿酒酵母Cdc73蛋白质为研究对象,首次解析了其C-端结构域结构。研究表明Cdc73的C-端氨基酸序列非常保守,其结构是一个由中心6个p折叠片被6个α螺旋包裹的α/β结构,通过Dali在线比对发现其与经典的GTPase的结构非常类似。但序列分析显示Cdc73与GTPase的同源度非常低,且关键区域也不保守。通过等温滴定量热法检测不到Cdc73与GTP明显结合,酶活实验也检测不到Cdc73具有水解GTP的GTPase酶活性,因此Cdc73尽管与GTPase具有类似的折叠方式,但可能不是一个GTP水解酶。该结构的解析为加深人们对Pafl复合物组装的认识提供了重要思路,为Cdc73与RNA Pol Ⅱ相互作用的研究以及人源的Cdc73与疾病相关研究等提供了重要的结构基础。
     DNA是生命遗传信息的载体,各种内源性或者外源性的DNA损伤因素会使其受到不同程度的损伤,从而导致基因组不稳定或基因组重排,进而导致一些疾病的发生。DNA损伤包括单链DNA损伤和双链DNA损伤。针对不同的DNA损伤。细胞内有许多不同的修复方式对其进行修复,包括碱基切除修复、核苷酸切除修复、错配修复、同源重组修复等。除了这些精确修复外,还有另外一种在复制叉延滞时进行的绕过损伤的修复方式,叫DNA损伤容限(DNA Damage tolerance, DDT),也叫复制后修复(PRR)。在复制后修复中有一种是无错修复方式(error-free),该过程和同源重组方式很类似,需要Rad51蛋白的参与。酿酒酵母Shu复合物由Csm2、Psy3、Shul和Shu2四个亚基组成,其通过抑制Srs2的活性促进Rad51依赖的同源重组发生。酿酒酵母中Psy3、Shul、Shu2在人源中对应的同源蛋白分别是Rad51D、Xrcc2和Sws1,其中裂殖酵母中Shu2的同源蛋白Sws1与Srs2有直接相互作用。最近发现人源中的Sws1能与一个新发现的蛋白质Swsap1形成稳定的复合物并参与同源重组修复过程。为理解该复合物的形成及相互作用机制,本文第二部分致力于解析人源Sws1-Swsap1复合物的结构。
Pafl complex which contains Pafl, Cdc73, Ctr9, Leol and Rtfl (recently discovered hSki8is one subunit in human Pafl complex) subunits is a conserved complex in eukaryotes. The main function of Pafl complex is taking part in transcription including initiation, elongation and termination together with RNA pol II. As one of the first identified subunits of PaflC, yeast Cdc73(yCdc73) takes part in many transcription-related processes, including binding to RNA polymerase II, recruitment and activation of histonemodification factors and communication with other transcriptional activators. The human homologue of yCdc73, parafibromin, has been identified as a tumour suppressor linked to breast, renal and gastric cancers.
     In this work, we targeted yeast Cdc73for structural studies and determined the C-terminal structure of yCdc73. The C-terminal of Cdc73is very conserved and a DALI server search indicates the C-terminal structure of Cdc73is a canonical a6/p6GTPase-like fold, although the primary sequence of it has very low identity to GTPases and is not conservative at the key region. No significant GTP-binding ability and hydrolysis activity were detected by ITC and enzyme activity assay. So Cdc73, with a GTPase-like fold, may not be a GTPase. The determination of the structure of Cdc73has provided further insights into the assembly way of Pafl complex and the structural basis for study on Cdc73-RNA PolⅡ interaction and human related diseases.
     DNA, the carrier of genetic information, is always induced a wide range of lesions by exogenous or endogenous DNA damaging agents. The genome will be instabile or re-arrange if the lesions of DNA are not repaired, and then some related diseases will occur. There are many DNA repair pathways to different DNA lesions, including base excision repair, nucleotide excision repair, mismatch repair and homologous recombination repair. In addition to these accurate repair pathways, when presence of replication-blocking lesions, there is another DNA damage tolerance (DDT) mechanism which does not remove DNA damages in the template strand, but bypass the DNA lesions during replication to prevent replication fork collapse, which is also named post-replication repair (PRR). An error-free pathway within PRR is very similar to the Rad51dependent homologous recombination repair (HRR). The yeast Shu complex is comprised of four subunits, Csm2, Psy3, Shu land Shu2, which promotes Rad51dependent homologous recombination via inhibit the activity of Srs2. The human orthologous of yeast Psy3, Shul and Shu2are Rad51D, Xrcc2and Sws1respectively. Sws1, the Shu2orthologous in fission yeast, was reported to have the ability to interact with Srs2. A newly identified human Swsl associate protein, Swsapl can form a stable complex with Swsl and takes part in homologous recombination repair. We are trying to solve the structure of Swsl-Swsapl complex and explain its function mechanism.
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