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Biotechnology with a pinch of salt - isolation of DNA repair enzymes from halophilic archaea

少许盐的生物技术——从嗜盐古菌中分离 DNA 修复酶

基本信息

批准号：
1645070
负责人：
金额：
--
依托单位：
University of Nottingham
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1645070
关键词：
Biotechnology pinch salt isolation DNA

项目摘要

BackgroundHel308 is a monomeric 3' to 5' DNA helicase conserved throughout archaea and metazoans however, is absent from bacteria and fungi. Hel308 is a member of the helicase superfamily 2. An investigation into Drosophila melanogaster mutants which exhibited hypersensitivity to DNA crosslinking agents led to the discovery of Hel308 and consequently the human homologue HELQ. Dependant on the organism of study, Hel308 is known by other names including HELQ in higher eukaryotes, and Hjm in some archaeal species for example Purococcus furiosus and Sulfolobus tokodaii. Though a member of the Ski2 family of helicases, Hel308 acts analogously to RecQ DNA family helicases and has consequently been implicated in a variety of overlapping DNA repair pathways, as well as homologous recombination, and genome stability. Despite these connections, the exact role of Hel308 has remained elusive.OutlineStructural, biochemical, and genetic analyses of Hel308 and its respective gene, have started to unravel the Hel308 mystery. Utilizing both the biochemical and genetic tool-boxes of Haloferax volcanii, protein and genetic interacting partners of Hel308 will be further explored under both native and stress conditions. Additionally, mutations of key residues identified via structural and sequence analysis will be investigated for phenotypic consequences. Overall, the function(s) of and the regulatory mechanism(s) of Hel308 will be examined.Unfortunately, no crystal structure for the Haloferax volcanii Hel308 has been obtained to date, there are however resolved crystal structures of three Hel308 homologues. From these crystal structures Hel308 is shown to be composed of 5 domains around a central DNA binding core which is identifiable by a lining of essential DNA binding residues. The 3' ssDNA overhang threads through this DNA binding core and the five domains drive a 13-hairpin between the two strands subsequently separating the DNA duplex. As with all superfamily 2 helicases Hel308 requires ATP binding and hydrolysis to enable translocation along the DNA. Key residues targeted for mutation include Walker A and B ATPase dead mutations, both independently and in combination with the previously identified hyper-recombinant point mutations.It has recently been shown that in Haloferax volcanii, mutants deleted for radB exhibit a decrease in the level of homologous recombination to approximately 5% of that in wild type cells. However, a double deletion of hel308 with radB restores, in part, homologous recombination. This data suggests an anti-recombinase role for Hel308. Further genetic analysis between hel308 and the homologous recombination pathway will be conducted focussing on the relationship between RadA and Hel308.ImpactThe mammalian Hel308 homologue HELQ, has previously been implicated in genome stability upon the recognition of several certain Hel308 single nucleotide polymorphisms (SNPs) being more prevalent in numerous types of head and neck cancers including esophageal squamous cell carcinoma and gastric adenocarcinoma. In addition to this, it has been suggested that HELQ has a critical role in germ cell maintenance and tumour suppression of ovarian cancers in mammals, indicating a possible role in replication-coupled DNA repair. Due to the evolutionary descent of eukaryotes from archaea, archaea present an opportunity to explore the function of eukaryotic HELQ in a simplified system. By exploring the function of Hel308 in Haloferax volcanii, we can begin to elucidate the mechanism of HELQ and therefore understand better how HELQ affects genome stability and DNA repair. Ultimately this understanding could lead to improved treatments for these cancers.

Hel 308是一种在古生菌和后生动物中保守的单体3'到5' DNA解旋酶，但在细菌和真菌中不存在。Hel 308是解旋酶超家族2的成员。一项对黑腹果蝇突变体的研究发现了Hel 308和人类同源物HELQ，这些突变体对DNA交联剂表现出超敏反应。根据研究的生物，Hel 308被称为其他名称，包括高等真核生物中的HELQ，以及一些古细菌物种中的Hjm，例如Purococcus furiosus和Sulfolobus tokodaii。虽然Hel 308是Ski 2解旋酶家族的成员，但其作用类似于RecQ DNA家族解旋酶，因此与多种重叠DNA修复途径以及同源重组和基因组稳定性有关。尽管有这些联系，Hel 308的确切作用仍然难以捉摸。概述Hel 308及其相应基因的结构，生物化学和遗传分析，已经开始解开Hel 308之谜。利用Haloferax volcano ii的生物化学和遗传工具箱，将在天然和胁迫条件下进一步探索Hel 308的蛋白质和遗传相互作用伴侣。此外，将研究通过结构和序列分析鉴定的关键残基突变的表型后果。不幸的是，迄今为止还没有获得Haloferax volcanii Hel 308的晶体结构，但是有三个Hel 308同系物的解析晶体结构。从这些晶体结构中，Hel 308显示出由围绕中心DNA结合核心的5个结构域组成，所述中心DNA结合核心可通过必需DNA结合残基的衬里来识别。3' ssDNA突出端穿过该DNA结合核心，并且五个结构域驱动两条链之间的13-发夹，随后分离DNA双链体。与所有超家族2解旋酶一样，Hel 308需要ATP结合和水解以使得能够沿着DNA移位。靶向突变的关键残基包括步行者A和B ATP酶死亡突变，无论是独立的还是与先前鉴定的超重组点突变相结合。最近已经表明，在Haloferax volcano ii中，缺失radB的突变体表现出同源重组水平下降至野生型细胞中同源重组水平的约5%。然而，hel 308与radB的双缺失部分恢复同源重组。该数据表明Hel 308具有抗重组酶作用。将对hel 308和同源重组途径之间进行进一步的遗传分析，重点关注RadA和Hel 308之间的关系。影响哺乳动物Hel 308同源物HELQ，在识别几种特定的Hel 308单核苷酸多态性（SNP）后，在包括食管鳞状细胞癌和胃腺癌在内的多种头颈部癌症中更为普遍。除此之外，已经表明HELQ在哺乳动物的生殖细胞维持和卵巢癌的肿瘤抑制中具有关键作用，表明在复制偶联DNA修复中可能起作用。由于真核生物是从古细菌进化而来的，古细菌为在简化的系统中探索真核生物HELQ的功能提供了机会。通过研究Hel 308在沃氏盐藻中的功能，我们可以开始阐明HELQ的作用机制，从而更好地理解HELQ如何影响基因组稳定性和DNA修复。最终，这种理解可能会导致这些癌症的治疗方法得到改善。