Molecular insights into ubiquitin remodelling factor p97(VCP) in DNA damage response and genome stability

Your body is built of billions of cells, and they all have to work as a team. Almost everything a cell does involves a protein. Proteins relay decisions, build structures, translate genetic instructions, repair damage, and carry signals. So it is not surprising that cancer is marked by proteins not working properly. Sometimes the proteins fail; sometimes they do their job too well, or do it at the wrong time.

Proteins are created and recycled constantly; this makes sure that proteins do not get old and damaged. The balance between making and recycling proteins is kept in tight check, making sure that the amount of a given protein is just right.

Our work focuses on the way cells recycle proteins. We know that proteins that are old or not needed are labelled for recycling with a tag called ubiquitin. Huge recycling machines called proteasomes grab hold of the ubiquitin handle and recycle the attached protein. We don't know in detail when and how the ubiquitin label is attached. We want to know how this works during the process of repairing damaged DNA.

Damaged DNA can be a starting point for cancer; but it's also the key to effective radio- and chemotherapy. If we knew how cells recycle the proteins that repair radiation and chemotherapeutic damage to DNA then we would be able to design ways to make radio- and chemotherapy more effective.This grant will fund research to improve our understanding of the proteins involved in DNA damage repair and the processes that recycle them.

Technical Summary

Our preliminary screen with siRNA against 13 members of UBX-family identified several candidates that strongly affect repair by homologous recombination, as measured by the DR-GFP reporter assay. We will establish which other p97-adapters are involved in processing of K48-ubiquitin signal at sites of DSB, and thus in DSB repair. We will use both laser micro-irradiation assays developed in our laboratory or ultrasoft X-ray (USX) micro-irradiation to induce DNA damage. This assay will screen for UBX-proteins that are relevant for the function of p97 at sites of DSB.

p97 is detected at sites of DSB after laser treatment, where it forms IR-dependent complex with RNF8 and K48-polyubiquitinated substrates. Now our goal is to understand the interactome of p97 and DSB related proteins. For this purpose we will isolate p97 complex from chromatin under different genotoxic conditions (e.g. exposure to IR or treatment with HU) and analyse p97 adapter interactome and substrate proteome by mass spectrometry. The complex formation between p97, its adapters and DSB repair proteins will be further analysed to define these interactomes and find out their cellular relevance.p97 together with its adapter DVC1 and E3 ubiquitin ligase RNF8 have an essential role in DNA replication and replication-related repair. We aim to understand how the p97-DVC1-RNF8 cascade orchestrates DNA replication. We will use standard techniques in the DNA replication field such as FACS analysis, pulsed-field gel electrophoresis, DNA fibre assays and confocal microscopy to establish the mechanism by which p97-DVC1-RNF8 regulates DNA replication and thus genome stability, and disorders such as Werner-like premature aging syndrome in human.