A protein called RNF4 acts as a molecular matchmaker, bringing together a damaged protein and a ubiquitin tag that marks it for repair or destruction. This research addresses a fundamental gap in understanding how cells protect their DNA from damage. When DNA breaks, cells must quickly repair it or risk mutations that can lead to cancer. RNF4 is known to be essential for this process, but exactly how it works—how it grabs the right proteins and attaches the right tags—remains unknown. The team will use proteomics to identify RNF4’s targets after DNA damage, and structural biology to capture the precise moment RNF4 transfers ubiquitin to its substrate. This is fundamental science. There is no immediate practical application. However, RNF4 is already known to be required for the therapeutic effect of arsenic trioxide in treating acute promyelocytic leukaemia. A detailed molecular understanding of how RNF4 works could eventually inform the design of drugs that mimic or block its activity, potentially leading to new cancer therapies. More broadly, understanding how cells maintain genome integrity is a core question in biology, and past discoveries in this area have underpinned major advances in cancer treatment and genetic medicine.
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RNF4 is a RING containing ubiquitin E3 ligase with specificity for polySUMO chains that is required for the therapeutic effect of arsenic trioxide in treatment of Acute Promyelocytic Leukaemia. Recently we established that RNF4 is required to maintain genome stability in higher eukaryotic cells. Our aim is to define the role of RNF4 in the DNA damage response and establish the molecular mechanism employed by RNF4, to catalyse transfer of ubiquitin to substrate. Our objectives are 1) to use quan titative proteomics to identify proteins that are selected as polySUMO modified substrates of RNF4 in response to DNA damage. 2) to establish how RNF4 recruitment leads to generation of a ubiquitin modification that is recognised by effector proteins that translate the ubiquitin signal into functional outputs. Importance of modifications will be determined by mutational analysis using in vivo readouts of DNA repair efficiency. Mechanisms by which RNF4 influences repair will be established using in vitro systems that recapitulate defined steps in repair. 3) Having recently proposed a model for the RING mediated transfer of ubiquitin to substrate we will test this using structural, biophysical and biochemical approaches and provide a description of the complex between ubiquitin loaded E2 and a dimeric RING ligase poised for catalysis. Ultimately our goal is to determine the structure of the complex containing polySUMO substrate, RNF4 E3 ligase and ubiquitin loaded E2. This would be a fir st in its class structure and would shed light on catalystic mechanisms employed by this important group of RING ligases.
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