Optimising translocation of Hel308 helicase for improved nanopore DNA sequencing

DNA解旋酶在DNA的复制、重组和修复中起着关键作用。Hel308是一种DNA解旋酶，它能够处理同源重组中出现的DNA结构，这是一种用于修复DNA双链断裂的保守机制。Hel308解旋酶存在于高等真核生物(称为Helq)和古生物中，但不存在于低等真核生物或细菌中。Hel308解旋酶被用于生物技术应用，它们帮助通过DNA测序流动细胞的纳米孔转移DNA。我们发现Hel308保守区域的氨基酸替换导致了体外高活性的DNA结合和DNA退火活性，并导致体内非交叉重组的大幅增加-但对细胞生长或DNA损伤的修复没有任何影响。解旋酶活性的这种变化与沿DNA移位的能力变化是一致的，并表明Hel308的这个保守区域可能是纳米孔DNA测序技术改进的关键。该项目的初始阶段将使用模型考古菌Haloferax Volcanii可用的一套广泛的遗传工具来筛选在体内显示变化活动的Hel308变体。基因筛查将被用来识别当发生突变时会导致解旋酶转位活性潜在变化的残基。例如，基因交换率的升高(或降低)预示着解旋酶易位的改变，并将通过高通量GFP-异种等位基因重组试验进行鉴定。该项目的这一部分将由Thorsten Allers教授领导。在项目的第二阶段，在基因筛查中确定的残留物将针对来自考古甲烷热营养杆菌的Hel308解旋酶中的氨基酸替换。来自这种模式古菌物种的蛋白质很容易进行生化分析。纯化的Hel308蛋白将在凝胶和单分子FRET分析中进行研究，以确定它们在体外改变的DNA结合和DNA退火活性。该项目的这一部分将由埃德·博尔特博士领导。在项目的最后阶段，将进一步调查Hel308名候选人的DNA易位活动。基于minion flow cell平台的单分子皮秒分辨率纳米孔镊子将用于监测单个Hel308酶沿着DNA模板的移位，从而确定特定序列的酶动力学。该项目的这一部分将与合作伙伴组织牛津纳米孔技术公司共同实施。

DNA helicases play key roles in the replication, recombination and repair of DNA. Hel308 is a DNA helicase that was identified by its ability to process DNA structures that occur in homologous recombination, a conserved mechanism that is used to repair DNA double-strand breaks. Hel308 helicases are found in higher eukaryotes (where they are known as HELQ) and in archaea, but not in lower eukaryotes or bacteria. Hel308 helicases are used in biotechnological applications, where they help to translocate DNA through the nanopore of DNA sequencing flowcells.We have found that amino acid substitutions in a conserved region of Hel308 lead to hyperactive DNA binding and DNA annealing activities in vitro, and result in vivo in a substantial increase in non-crossover recombination - but without any impact on cell growth or the repair of DNA lesions. Such changes in helicase activity are consistent with an altered ability to translocate along DNA, and indicate that this conserved region of Hel308 may hold the key to improvements in nanopore DNA sequencing technologies.The initial phase of the project will use the extensive set of genetic tools available for the model archaeon Haloferax volcanii to screen for variants of Hel308 that display altered activities in vivo. The genetic screen will be used to identify residues that when mutated, lead to potential changes in helicase translocation activity. For example, elevated (or reduced) rates of genetic exchange are indicative of altered helicase translocation, and will be identified via high-throughput GFP-heteroallele recombination assays. This part of the project will be led by Professor Thorsten Allers.In the second phase of the project, residues identified in the genetic screen will be targeted for amino acid substitutions in the Hel308 helicase from the archaeon Methanothermobacter thermautotrophicus. Proteins from this model archaeal species are tractable for biochemical analysis. Purified Hel308 proteins will be studied in gel-based and single-molecule FRET assays, to determine their altered DNA binding and DNA annealing activities in vitro. This part of the project will be led by Dr Ed Bolt.In the final phase of the project, Hel308 candidates will be further investigated for their DNA translocation activity. Single-molecule picometer-resolution nanopore tweezers, which are based on a MinION flowcell platform, will be used to monitor the translocation of individual Hel308 enzymes along a DNA template, and thereby determine sequence-specific enzyme kinetics. This part of the project will be carried out in conjunction with the partner organisation, Oxford Nanopore Technologies.