Nucleosome remodelling at sites of DNA damage: regulation and mechanism of the remodeller Fun30/SMARCAD1

• Applicant: Professor Dr. Boris Pfander
• Subject Area: Cell Biology
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 299273611

Eukaryotic DNA is packaged into chromatin and all cellular processes that deal with DNA are adjusted to this feature. While nucleosomes may interfere with DNA accessibility, creating a need for removal or repositioning, they are also being employed and modified by many cellular pathways to serve as local signalling molecules. Therefore, it is not possible to completely understand any DNA associated process without understanding the dynamic changes in chromatin that go alongside. The cellular machinery that mediates these changes can be classified into histone modifying enzymes and nucleosome remodellers, which catalyse the ATP-dependent change in nucleosome DNA contacts and/ or nucleosome composition.In case of the DNA damage response (DDR), significant progress has been made in understanding the role of nucleosome modifications, but the role of nucleosome remodellers is still poorly understood. A useful model system to study the role of nucleosome remodellers in the DDR are double-strand breaks (DSBs), which elicit substantial alterations in the surrounding chromatin and can be efficiently induced experimentally at specific sites in the genome. Using this system, a nucleosome remodeller named Fun30 has recently been identified as important novel factor in the DDR of budding yeast. Specifically, Fun30 has been shown to be critical for DNA end resection, an early nucleolytic DNA processing step that prepares the DSB for its repair by homologous recombination. It is however unclear how Fun30 DNA targeting and activity are regulated, and how Fun30-dependent nucleosome remodelling promotes DNA end resection and what its direct targets are.Our preliminary data suggests that Fun30 function at DSBs depends on a protein complex, in which Fun30 binds to the Dpb11 scaffold protein in a phosphorylation-dependent manner. We have generated a specific, Dpb11 binding-deficient mutant of Fun30, which shows defects in DNA end resection suggesting that Dpb11-binding is regulating Fun30 possibly by targeting Fun30 to DSBs. Starting from these findings we aim to reveal how Fun30 action at a DSB is regulated and by which mechanism Fun30-dependent nucleosome remodelling promotes DNA end resection. Furthermore, we will transfer our findings from the budding yeast model to higher eukaryotes and test whether the human Fun30 homolog SMARCAD1 is under similar control, given that it also promotes DNA end resection. We expect that the proposed strategy will not only allow us insights into the regulation and mechanism by which Fun30/SMARCAD1 promotes repair of DNA lesions, but that the identified principles and approaches may be translated to future studies on other nucleosome remodellers that act in the DNA damage response.

• DFG Programme: Research Grants