Mechanistic analysis of DNA helicases using nanopore-based single molecule assays

点击翻译按钮获取中文摘要

Next generation DNA sequencing methods such as those pioneered by Oxford Nanopore Technologies (ONT) haverevolutionised biology by allowing rapid DNA sequencing at the whole organism level and impacting fields as diverse asevolutionary biology, structural and molecular biology, agriculture, diagnostics and personalised medicine. The ONTtechnology works by coupling an ATP-dependent DNA motor protein to a nanoscale pore in a biological membrane. Asthe motor delivers DNA through the pore, the base composition is determined by changes in the current across themembrane ultimately yielding accurate long read DNA sequencing data. In addition to their core uses for long-read DNAsequencing and rapid diagnostics, nanopore-based assays have also found unexpected applications in basic science. Forexample, because nanopore DNA sequencing is rate-limited by the activity of the motor, the sequencing traces alsocontain rich information on the kinetics of DNA translocation providing unique insights into the mechanism(s) ofessential enzymes such as helicases. In this project, you will integrate new DNA helicases into nanopore-based DNA sequence devices with two major goals.Firstly, we aim to improve the speed, accuracy, simplicity and robustness of nanopore DNA sequencing by using DNAhelicases with novel and desirable biochemical properties. Secondly, we aim to study the mechanism of action of DNAhelicases of medical interest and how they are affected by small molecule drugs or genetic mutation. Importantly, weanticipate that nanopore traces will yield unprecedented detail on the nature of the individual steps that are made alongDNA and their chemo-mechanical coupling to ATP hydrolysis. Your project will be based in the laboratory of Prof. MarkDillingham in the DNA:protein interactions Unit at the University of Bristol but will also involve close collaboration withOxford Nanopore Technologies (1) including internships spent at their Oxford headquarters. The Dillingham lab (2) isstudying helicases involved in the repair of broken DNA, including bacterial enzymes that are considered attractivetargets for antibiotics and human enzymes implicated in genetic diseases including cancer. For further details andexamples of our recent work see our website or contact the laboratory (2). (1) https://nanoporetech.com/ (2) https://research-information.bris.ac.uk/en/persons/mark-s-dillingham