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Investigating the regulation of DNA non-homologous end-joining through Ku70/80 interacting factors
DNA double-strand breaks are the most deleterious type of DNA damage that cells experience,
which makes the study of double-strand break repair extremely important. Unrepaired or
aberrantly repaired DNA can result in changes to core genes with critical function and thus
lead to multiple diseases. Two main repair pathways for double-strand breaks exist:
homologous recombination (HR) and non-homologous end-joining (NHEJ). Whilst the
regulation of HR has been heavily investigated, the regulation of NHEJ remains to be fully
explored. The aim of this thesis is to investigate the regulation of DNA NHEJ through
interacting factors of the core NHEJ protein heterodimer, Ku70/80 (Ku).
This thesis consists of three main research projects. The first, explores the potential role of
the CUL4 substrate adaptor, WDR76, in the removal of Ku from sites of DNA damage. Data
presented here highlight a role of WDR76 in the DNA damage response (DDR), and through
effects on Ku removal kinetics, suggest a role for WDR76 in the regulating NHEJ.
The second research project investigates a potential cyclin-dependent kinase
phosphorylation site on the protein paralog of XRCC4 and XLF (PAXX). As PAXX is a Ku
interactor with a role in NHEJ, the effect of PAXX phosphorylation is investigated as a potential
NHEJ regulatory system.
Lastly, I investigate the role of the RecQ helicase WRN, whose precise roles in the DDR are
unclear. As an interactor of both HR and NHEJ proteins, WRN may affect the regulation of
both pathways. WRN knockout cells were generated and a CRISPR-Cas9 screen performed to
identify suppressors of WRN sensitivity to DNA damage. The targets identified offer insights
into WRN function.BBSRC
Horizon Discovery
Steve Jackson Laborator
Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination.
Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.Cancer Research UK (Grant IDs: C6/A18796, C6946/A14492, C6/A18796), European Research Council (Grant ID: 310917), Wellcome Trust (Grant ID: WT092096), University of Cambridge, Institut PasteurThis is the final version of the article. It first appeared from Elsevier (Cell Press) via http://dx.doi.org/10.1016/j.celrep.2016.08.06