Understanding DNA helicases involved in replication-repair mechanisms

Supervisors: Carlos Penedo, Bela Bode

Project Description:
Chemical damage and physical barriers to DNA synthesis at replication forks need to be overcome to ensure genome duplication. Replication-repair enzymes that reactivate blocked or stalled replication forks are thus crucial for health by preventing genome instability. The human helicase HelQ is a ssDNA ATPase that translocates with 3’ to 5’ directionality and efficiently separates DNA strands as a helicase. Although HelQ is an essential enzyme required for repair of replication-blocking lesions and HelQ mutations are known to cause slowed and collapsed replication, its structure and function is still poorly understood. Open questions remaining include: what is the oligomeric structure of HelQ?, how does this relate to its translocation mechanism and to how it physically interacts with other recombination proteins?

We have recently initiated a collaboration with Dr Edward Bolt at the University of Nottingham (School of Life Sciences) to unveil the biochemistry and biophysics of HelQ, Hel308 (an archaeal homologue) and PolQ helicases [1] during DNA replication-repair. In this project, we combine the expertise in Electron Paramagnetic Resonance (EPR,) from Dr Bela Bode’s group (School of Chemistry, St Andrews) with Carlos Penedo’s group (School of Biology and School of Physics and Astronomy, St Andrews) experience in single-molecule technologies to answer these questions. EPR-based techniques will be used to determine the oligomeric state and the structure of these proteins in solution. On the other hand, single-molecule fluorescence resonance energy transfer (FRET) microscopy will be used to characterize the translocation dynamics of HelQ in intact and blocked substrates. For this, we have a range of single-molecule microscopes with temporal resolutions from milliseconds to minutes. Hybrid microscopies combining fluorescent detection with mechanical stretching and torsional manipulation of the DNA substrate using magnetic tweezers are also available. The Bode and Penedo groups have a significant track record of collaboration studying DNA-protein interactions [2].

The project is interdisciplinary and merges components of molecular biology, structural biology and biophysics. Through the course of these studies the student will receive training in molecular biology, biochemistry and cutting edge EPR and single-molecule biophysical techniques, thus providing him/her with excellent skills and a broad knowledge base to be well positioned for a future career in biotech, the pharmaceutical industry or academia.


[1] DNA binding and unwinding by Hel308 helicase requires dual functions of a winged hélix domain. S. J. Northall, R. Buckley, N. Jones, J. C. Penedo, P. Soultanas, E. L. Bolt. (2017) DNA Repair 57, 125-132

[2] Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multiprocess approach. Michael J. Morten, Jose R. Peregrina, Bela E. Bode, Malcolm F. White* and J. Carlos Penedo*, (2015) Nucleic Acids Res. 43, 107-10924