Mechanisms underlying the function of XLF in DNA double-strand break repair

DNA-reparasjon opprettholder den genomiske stabiliteten til cellene våre, som utfordres av eksterne og interne, fysiske og kjemiske faktorer. Når DNA-reparasjonsgener er mutert, kan ikke korrespondentfaktorer og molekylveier fungere ordentlig. Det forårsaker en rekke lidelser, alt fra immunsvikt, abnormiteter i nervesystemet og kreft. XLF er DNA-reparasjonsfaktoren, og mutasjoner i XLF-genet fører til immunsvikt og hjerneavvik hos mennesker. Mekanismer som ligger til grunn for funksjonen til XLF er ikke klare ennå, og vi foreslo den tverrfaglige tilnærmingen for å belyse XLFs rolle i DNA-reparasjon og i beskyttelse mot lidelser. Nylig fant vi ut at funksjonen til XLF delvis oppveies av andre DNA-reparasjonsfaktorer, DNA-PKcs, Paxx og Mri/Cyren. I løpet av 2016-2019 (dette prosjektet) utviklet og karakteriserte vi to nye musemodeller som mangler enten PAXX eller Mri. Disse dataene er delvis publisert i internasjonale tidsskrifter, DNA-reparasjon, FEBS åpen bio og Biomolekyler. Totalt sett antyder funnene våre flere nye kandidatgener for prognosen for immunsvikt hos mennesker.

Primary immunodeficiency needs to be rapidly diagnosed and treated in children, otherwise, it can result in death. To determine the impact of new DNA repair factors on immune system development, we generated, characterized and published two mouse models, and multiple genetically modified cell lines. These models are available for researchers worldwide, e.g. mice lacking PAXX or MRI have been transferred to Oslo. Our results established roles for several DNA repair factors, including PAXX, Mri, MDC1, Gcn5, and PCAF in lymphocyte development. This knowledge can be translated to clinics for a more precise diagnosis of primary immunodeficiency. Moreover, one PhD candidate and ten MSc students were trained during this project. This positively influences society by providing experts in immunology and biomedical research.

In this project, I will use a multi-disciplinary approach combining cell biology, mouse genetics and proteomics to elucidate new aspects of the non-homologous DNA end joining (NHEJ) pathway. NHEJ repairs DNA double strand breaks (DSBs) to maintain genome stability and defects in the pathway cause immunodeficiency and neurological diseases. The NHEJ pathway includes Ku70/Ku80 (Ku), DNA-PKcs kinase, and XRCC4/Ligase4 complex that ligates two DSB ends together. XRCC4-like factor (XLF) is a protein that directly associates with XRCC4. Deficiency of XLF leads to genomic instability and results in microcephaly and immunodeficiency in humans. However, the exact function of XLF is unclear. Deficiencies of Ku, XRCC4, and Ligase4 abrogate NHEJ, while inactivation of XLF or DNA-PKcs leads to modest defects in DSB repair. Mice deficient in either XLF or DNA-PKcs are alive and show mild NHEJ defects; surprisingly, combined deficiency of XLF and DNA-PKcs leads to perinatal lethality associated with a complete loss of NHEJ. My preliminary data indicate that deletion of Ku completely rescues the lifespan of these double deficient mice, pointing to a toxic effect of Ku in the absence of both XLF and DNA-PKcs. To explain this remarkable genetic rescue, I propose that in the absence of DNA-PKcs, XLF associates with other proteins and facilitates the removal of Ku from DSBs during DNA repair. To test this hypothesis, I will investigate the role of XLF in recruitment of Ku70 and Ligase4 to DSB sites with live-cell imaging and perform quantitative proteomics to identify XLF-associated proteins. Finally, I will use mouse genetics to determine whether the perinatal lethality of XLF/DNA-PKcs mice is a consequence of p53-dependent apoptosis and perform histopathology analyses to elucidate previously underappreciated roles for DNA-PKcs and XLF in mouse development. The scientific understanding of XLF function can lead to new therapeutic strategies as well as prognostic markers for disease