DNA break repair pathway choice: mechanism, control, bypass

• Applicant: Professor Dr. Boris Pfander
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 466479039

A DNA double strand break (DSB) is not only a severe form of DNA damage that threatens genome integrity, it is also a key molecular intermediate of genome editing procedures with high importance for biotechnology. To repair DSBs, eukaryotes can utilize one of several DSB repair mechanisms, including homologous recombination. Usage of different repair mechanism will lead to genetically different products of repair. Despite the critical dependence of genome editing technology on this cellular DSB repair decision, our understanding of how cells choose a specific DSB repair mechanism is still incomplete. Past research has revealed that DSB repair pathway choice is made during DNA end resection, the first step of homologous recombination, and that resection is in turn controlled by the cell cycle as well as the DSB-surrounding chromatin. However, we do not know all controls of resection and not in sufficient mechanistic detail. Therefore, efforts to manipulate the DSB repair decision from the outside and to make HR the default DSB repair pathway have had limited success so far. Here, we propose to biochemically reconstitute DNA end resection using purified proteins and on a defined chromatin substrate. We will use this in vitro system to test by which mechanism known resection regulators such as the cell cycle kinase CDK, the nucleosome remodeller Fun30/SMARCAD1 or the nucleosome binder Rad9/53BP1 act in order to promote or inhibit resection. Availability and previous characterization advise us to initially focus on budding yeast proteins. However, we will also take first steps into the human system focussing on the interplay between the two resection promoting factors, the tumor suppressor and nucleosome-targeting ubiquitin ligase BRCA1 and the nucleosome remodeller Fun30/SMARCAD1.A detailed understanding of resection control is necessary if we want to influence DSB repair pathway choice from the outside and advance genome editing procedures. Therefore, in the second part of the project, we aim to generate genetic conditions that allow for a synthetic bypass to the control of DNA end resection. Towards this goal, we will focus on a new layer of regulation by the cell cycle kinase DDK, which was recently identified as part of our preliminary work and could be the missing cornerstone in the regulatory framework of DSB repair pathway choice. We will attempt to bypass this new DDK-dependent control together with the already established CDK-dependent control mechanisms and test whether this enables us to synthetically activate homologous recombination throughout all cell cycle phases.

• DFG Programme: Research Grants