You will perform carbene footprinting (see Nature Communications., 2016, 7:13288) on recombinantly expressed and purified human RPA and HelQ proteins to map their sites of interaction. The methodology, developed in our lab, allows protein interaction sites to be identified through masking effects on a protein labelling by a photochemically activated probe. Labelling will be performed on each protein individually, then on the complex. Mass spectrometry will be used to map the sites of labelling and quantify any masking caused by formation of the complex. This will allow identification of the binding site on each protein.
In human cells DNA repair of blocked or broken DNA replication forks is essential for preventing genome instability that triggers cancers and premature aging syndromes. HELQ is important for DNA repair, highlighted by multiple effects of HELQ gene loss, including elevated mutation rates, increased cancer incidence and sterility. Genetic analyses have led to models implicating HELQ in controlling and limiting homologous recombination when DNA replication is being repaired, so that replication can resume without genetic rearrangements occurring. But biochemical mechanisms for how HELQ helicase contributes to these events are unknown. HELQ is an ATP-dependent DNA helicase that translocates single stranded DNA (ssDNA) with 3’ to 5’ polarity.
Recent work in our laboratories has revealed that purified human HELQ protein interacts with an essential human DNA replication/repair protein RPA, and in so doing this converts HELQ into a “super-helicase” that can disrupt DNA-protein barriers (Jenkins et al, manuscript in preparation for submitting to Nucleic Acids Res.). The central hypothesis leading to this project is that HELQ-RPA helicase complex specifically targets protein-DNA recombination complexes for removal to control their function and therefore control DNA repair in human cells.
We have already established that RPA and HELQ interact to form a stable complex detectable by gel electrophoresis. We will use recombinantly expressed and purified human RPA and HELQ proteins to achieve three objectives:
The University of NottinghamUniversity Park
Nottingham, NG7 2RD
Tel: +44 (0) 115 8466946
Connect with the University of Nottingham through social media and our blogs.
Campus maps | More contact information | Jobs
Browser does not support script.