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Investigating the importance of protein turnover in the DNA Damage Response.

Ionising Radiation-induced DNA double strand break repair orchestrated by the ubiquitin-dependent chaperon p97/VCP

My group has been addressing some of the fundamental biological questions relevant for DNA replication and repair, genome stability, oncology, radiation biology and cancer therapy (Figure 1). The overarching goal of the Ramadan laboratory is to elucidate the role of the ubiquitin-chaperon p97/VCP (p97) in two basic biological processes: chromatin-associated protein degradation (CHROMAD) and DNA-protein crosslink (DPC) proteolysis in genome stability and cellular response to radio- and chemo-therapy. We use DNA double strand break repair and DNA replication as the model systems to address our questions. p97 is an AAA+ ATPase that together with its cofactors, associated E3-ubiquitin ligases and deubiquitinating enzymes forms the p97 system, which plays one of the central roles in the ubiquitin system and protein homeostasis. Thus, the p97 system contributes to both physiological (e.g., genome stability) and pathological processes such as cancer cell survival and progression.

Figure 1: The multiple roles of the p97 system on chromatin. This cartoon summarises all the discoveries of our laboratory in the field of p97-dependent chromatin degradation and proteolysis. The p97 system is composed of ATPase p97 and its cofactors (so far 40 known proteins, but this list is growing). p97-cofactors contain p97-Binding Domains and majority of them also contain Ubiquitin Binding Domains. Thus, p97-cofactors bridge the ATPase p97 to a specific, mostly ubiquitinated, substrate. Once recruited to a specific substrate on chromatin, p97 employs its ATPase activity to remodel - unfold and/or extract - the substrate. Finally, the remodelled substrate is removed from chromatin and degraded or recycled. To date, it is not known how many different p97 complexes exist and what are their substrates. Importantly, as p97 is ATPase and some of the p97-cofactors possess enzymatic activities (such as the E3-ubiquitin ligase activity, deubiquitinating activity, or protease activity), the p97 system is druggable at various levels, representing therefore endless opportunities for cancer therapyFigure 1: The multiple roles of the p97 system on chromatin. This cartoon summarises all the discoveries of our laboratory in the field of p97-dependent chromatin degradation and proteolysis. The p97 system is composed of ATPase p97 and its cofactors (so far 40 known proteins, but this list is growing). p97-cofactors contain p97-Binding Domains and majority of them also contain Ubiquitin Binding Domains. Thus, p97-cofactors bridge the ATPase p97 to a specific, mostly ubiquitinated, substrate. Once recruited to a specific substrate on chromatin, p97 employs its ATPase activity to remodel - unfold and/or extract - the substrate. Finally, the remodelled substrate is removed from chromatin and degraded or recycled. To date, it is not known how many different p97 complexes exist and what are their substrates. Importantly, as p97 is ATPase and some of the p97-cofactors possess enzymatic activities (such as the E3-ubiquitin ligase activity, deubiquitinating activity, or protease activity), the p97 system is druggable at various levels, representing therefore endless opportunities for cancer therapy

 

Over the past decade the work of the Ramadan group (Figure 1) has directly demonstrated that p97-dependent protein disassembly and degradation on chromatin is essential for genome stability. However, it is not known how many different p97 complexes exist on chromatin, what is the composition of p97’s cofactors and substrates and how exactly different p97 complexes regulate DNA replication and DNA damage response after IR. By investigating the molecular mechanisms of the p97 system in DNA replication and DNA damage response after IR, this research programme aims to understand how protein turnover and degradation regulate genome stability, thus protecting cells from tumorigenesis and accelerated ageing. As the components of the p97 system emerge as druggable targets for cancer therapy and radiosensitisers, our ultimate goal is to identify a strong translational potential between our discoveries and cancer diagnosis and therapy. We are mostly using biochemical, cell and molecular biological approaches to address our questions.