Cryo-EM studies of DNA damage response proteins
EMSL Project ID
51280
Abstract
DNA double-strand breaks (DSBs) are toxic lesions associated with cancer. In chromatin, a complex of DNA and histone proteins that forms nucleosomes, DSBs can be repaired by two different pathways called non-homologous end joining (NHEJ) and homologous recombination (HR). To complement the extensive biochemistry studies that we have done so far, we will use cryo-electron microscopy (cryo-EM) to explore how histone ubiquitylation in response to DSBs triggers the chromatin recruitment of DNA damage response (DDR) proteins involved in HR DNA repair. We have initiated this project by developing a procedure for reconstituting the highly purified ubiquitylated nucleosome core particle (NCP-ub). We are requesting access to the Pacific Northwest Cryo-EM Center (PNCC) with the goal of determining the three-dimensional structures of NCP-ub in complex with DDR proteins. To illustrate feasibility of our proposed studies, we present preliminary cryo-EM data on a complex of NCP-ub and a domain from the ubiquitin ligase RAD18. With cryo-EM data recorded at PNCC, we will probe the NCP recognition mechanism of RAD18 and other DDR proteins involved in HR DNA repair. These cryo-EM structures will reveal the DSB recruitment mechanisms of RAD18 and other DDR proteins and provide a rational basis for understanding their modes of action in cells. In the long term, this knowledge could be harnessed for designing HR DNA repair inhibitors to potentiate the killing of cancer cells by poly (ADP-ribose) polymerases inhibitors (PARPi) and counteract resistance to PARPi.
Project Details
Start Date
2020-02-15
End Date
2021-03-17
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members