Electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry of DNA damage repair complexes
Group Leader page
ATM and ATR are members of the phosphoinsositide-3-kinase-like protein kinases family (PIKKs). These enzymes are activated in response to DNA damage and they are master kinases linking DNA damage to cell cycle progression and cell death. The enzymes are part of complexes that sense DNA breaks and phosphorylate a wide range of targets in the DNA damage response. Cancer cells rely on this pathway to survive genomic instability, and this has led to an interest in inhibiting these enzymes as a cancer therapeutic strategy.
The project involves understanding how these kinases are activated in response to DNA damage, with an emphasis on the interactions that they make with protein complexes on DNA. The project involves electron cryo-microscopy and hydrogen/deuterium exchange mass spectrometry of large enzyme complexes. The work builds on the experience of the group with other members of the PIKK family and the large-scale mammalian cell culture that has been optimised in the group for these enzymes. The structural work will be carried out in parallel with mammalian cell biology and in vitro enzymology. The hydrogen/deuterium exchange mass spectrometry will provide insight into the interactions that the enzyme complexes make in DNA repair. This technique has been optimised in the group to provide single-residue resolution.
Structural work will take advantage of the extensive electron cryo-microscopy facilities in the LMB.
Baretić, D. and Williams, R.L. (2014)
PIKKs--the solenoid nest where partners and kinases meet.
Curr. Opin. Struct. Biol. 29, 134–142.
Baretić, D., Berndt, A., Ohashi, Y., Johnson, C.M. and Williams, R.L. (2016)
Tor forms a dimer through an N-terminal helical solenoid with a complex topology.
Nat Commun 7, 11016.
Vadas, O. and Burke, J.E. (2015).
Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange-MS (HDX-MS).
Biochem. Soc. Trans. 43, 773–786.