The classical non-homologous end-joining (c-NHEJ) pathway is largely responsible for repairing DNA double-strand breaks (DSBs) in mammalian cells. Absence of c-NHEJ causes genomic instability in mice and leads to deficiencies in V(D)J recombination. XLF, (the last bona fide c-NHEJ factor discovered) is known to stimulate ligation by the core ligation complex: XRCC4-Ligase 4. However, the precise mechanism by which XLF stimulates XRCC4-Ligase 4 mediated DNA ligation is not well understood. Recent structural studies have shown that XLF can interact with XRCC4 to form filaments of alternating XRCC4 and XLF dimers; these filaments mediate DNA end bridging in vitro, providing a potential mechanism by which XLF might stimulate ligation. Here, we show that disrupting the interaction between XRCC4 and XLF by XRCC4 mutation, thereby abolishing filament formation, affects V(D)J recombination in cells and hinders the ability of cells expressing these mutants to survive in response to zeocin, a radiomimetic drug. Furthermore, we characterize an XLF mutant (L115A) that does not interact with XRCC4, and thus does not form filaments or bridge DNA in vitro. However, this mutant is fully sufficient in stimulating ligation of either blunt or cohesive DNA ends by X4/Lig4 in vitro. This separation of function mutant fully complements the zeocin sensitive phenotype and V(D)J recombination deficits of some XLF deficient cell strains but not others, suggesting a variable requirement for DNA bridging in different cell types. To determine whether lack of XRCC4/XLF bridging might be compensated for by other factors, candidate repair factors were disrupted in XLF or XRCC4 deficient cells. Loss of either ATM or the newly described XRCC4/XLF like factor, PAXX accentuates the cellular requirement for XLF. In the case of ATM/XLF loss, the increased cellular requirement can be attributed to its bridging function; however in case of PAXX/XLF loss the increased requirement for XLF is independent of bridging.
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