Optimising translocation of Hel308 helicase for improved nanopore DNA sequencing
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
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 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.
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.
IResidues 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.
Finally, 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.
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.
IResidues 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.
Finally, 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.
People |
ORCID iD |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/T008369/1 | 30/09/2020 | 29/09/2028 | |||
| 2886325 | Studentship | BB/T008369/1 | 30/09/2023 | 29/09/2027 |