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

背景：hel308是一种在古细菌和后生动物中保守的单体3′至5′DNA解旋酶，但在细菌和真菌中不存在。Hel308是解旋酶超家族2的成员。对对DNA交联剂过敏的果蝇突变体的研究导致了Hel308的发现，从而发现了人类的同源物HELQ。根据所研究的生物，Hel308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。Hel308虽然是Ski2解旋酶家族的成员，但其作用类似于RecQ DNA解旋酶家族，因此涉及多种重叠DNA修复途径，以及同源重组和基因组稳定性。尽管存在这些联系，但Hel308的确切作用仍然难以捉摸。对Hel308及其相应基因的结构、生化和遗传分析已经开始揭开Hel308的神秘面纱。利用火山盐铁ax的生物化学和遗传工具箱，在自然和胁迫条件下进一步探索Hel308的蛋白质和遗传相互作用伙伴。此外，通过结构和序列分析鉴定的关键残基突变将被研究表型后果。总的来说，我们将研究Hel308的功能和调控机制。不幸的是，迄今为止还没有获得火山盐藻Hel308的晶体结构，但有三个Hel308同源物的晶体结构。从这些晶体结构中可以看出，Hel308是由围绕一个中心DNA结合核心的5个结构域组成的，这个中心DNA结合核心可以通过一层基本DNA结合残基来识别。3' ssDNA悬垂线穿过DNA结合核心，5个结构域驱动两条链之间的13发夹，随后分离DNA双链。与所有超家族2解旋酶一样，Hel308需要ATP结合和水解才能沿着DNA进行易位。突变的关键残基包括Walker A和B ATPase死亡突变，它们可以独立存在，也可以与先前鉴定的超重组点突变结合使用。最近的研究表明，在火山盐黄貂中，因radB而缺失的突变体表现出同源重组水平下降到野生型细胞的约5%。然而，hel308与radB的双重缺失部分恢复了同源重组。这一数据表明Hel308具有抗重组酶的作用。进一步对hel308与同源重组通路进行遗传分析，重点研究RadA与hel308的关系。哺乳动物Hel308同系物HELQ先前已被认为与基因组稳定性有关，因为在许多类型的头颈部癌症（包括食管鳞状细胞癌和胃腺癌）中识别出某些Hel308单核苷酸多态性（SNPs）更为普遍。除此之外，有研究表明HELQ在哺乳动物生殖细胞维持和卵巢癌肿瘤抑制中起关键作用，这表明它可能在复制偶联DNA修复中起作用。由于真核生物从古细菌进化而来，古细菌提供了一个在简化系统中探索真核生物HELQ功能的机会。通过探索Hel308在火山盐藓中的功能，我们可以开始阐明HELQ的机制，从而更好地了解HELQ如何影响基因组稳定性和DNA修复。最终，这种认识可能会改善这些癌症的治疗方法。

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.