XRCC4's interaction with XLF is required for coding (but not signal) end joining

Abstract

XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together—stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

“…It also is possible that XRCC4/XLF bridging occurs prior to DNA-PK assembly; in fact, it is known that XLF requires Ku (but not DNA-PKcs) to be appropriately targeted to DNA damage in living cells (65,66). Our previous studies suggest that DNA-PK phosphorylation of XRCC4/XLF disrupts filaments (29). Work is ongoing to define how XRCC4/XLF filaments promote repair in living cells.…”

“…Immunoblotting was performed as described previously (29). Antibodies used in this study are rabbit polyclonal anti-XLF (Abcam), rabbit polyclonal anti-XRCC4 (Abcam), goat anti-C9ORF142 (Santa Cruz Biotechnology) for PAXX, rabbit anti-ATM (Serotec), and rabbit polyclonal anti-DNA-PKcs-phospho-S2056 (Abcam).…”

Functional overlaps between XLF and the ATM-dependent DNA double strand break response

“…In biochemical assays, XLF also enhances efficiency of the XRCC4/Lig4 complex to ligatelinearized plasmids in vitro [128]. Notably, non-lymphoid cells that express mutant forms of XRCC4 that are unable to interact with XLF carry out joining of SEs, but not CEs, in the context of extra-chromosomal V(D)J recombination substrates [129], suggesting potential functional compensation for XLF-XRCC4 end-bridging by RAG or other factors in the post-cleavage synaptic complex for joining of SEs [121, 129, 130]. …”

“…As mentioned above, one such factor might be RAG, which holds CEs and SEs in a post-cleavage synaptic complex and directs C-NHEJ-mediated joining of two CEs and two SEs, respectively, to each other [131, 132]. In this context, SEs are held more tightly than CEs in the RAG post-cleavage synaptic complex [133]; which in the context of proposed XLF synapsis functions could explain the lack of impact on SE joining versus CE joining when XRCC4-XLF interaction is disrupted [129]. If RAG does function redundantly with XLF during V(D)J recombination in developing lymphocytes, but not in non-lymphoid cells, it is conceivable that normal physiological V(D)J recombination may have evolved to optimize ability of RAG, for example via post-translational modifications, to hold CEs and/or SEs in post-cleavage synaptic complexes for proper end-joining and/or to contribute to C-NHEJ factor recruitment [12, 131, 132, 134].…”

Nonhomologous end joining: A good solution for bad ends

“…DNA-PKcs also phosphorylates the ligase complex – most prominently XRCC4 [148] and XLF [149]. The latter phosphorylations serve to reduce DNA binding and progressively disrupt the XRCC4-XLF-ligase IV filament [150], suggesting possible intermediate states where the filament is more flexible or allows greater access for processing factors.…”