Abstract
XRCC4 and DNA Ligase 4 (LIG4) form a tight complex that provides DNA ligase activity for classical non-homologous end joining (the predominant DNA double-strand break repair pathway in higher eukaryotes) and is stimulated by XLF. Independently of LIG4, XLF also associates with XRCC4 to form filaments that bridge DNA. These XRCC4/XLF complexes rapidly load and connect broken DNA, thereby stimulating intermolecular ligation. XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM. Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues. These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes, but without affecting their ability to stimulate LIG4 activity. Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments.
Highlights
In mammalian cells, classical non-homologous end joining (c-NHEJ) is the primary pathway that repairs DNA double-strand breaks (DSBs)
This intrinsic XLF DNA bridging activity is strongly stimulated by XRCC4 (Figure 2G, lanes 3 and 7) in a manner that depends on physical interaction between XLF and XRCC4, as tested using an XLF mutant (L115D) described previously that does not interact with XRCC4 but has an intact C-terminal tail (Figure 2G, lanes 4 and 8, Malivert et al, 2010; Roy et al, 2015)
We propose that ATM/DNA-PK phosphorylation of XRCC4/XLF filaments facilitates their dissociation from DNA
Summary
Classical non-homologous end joining (c-NHEJ) is the primary pathway that repairs DNA double-strand breaks (DSBs). This pathway is specialized in the repair of two-ended DSBs like those that are induced by ionizing radiation or by many radiomimetic drugs. Core c-NHEJ effectors include the heterodimeric Ku DNA end sensor, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), DNA Ligase 4 (LIG4), and the structurally related XRCC4 and XLF proteins. Current dogma advocates a sequential model for c-NHEJ as follows: (1) Ku performs the recognition step, rapidly and avidly binding and protecting broken DNA ends; (2) Ku recruits DNA-PKcs and promotes DNA end synapsis, regulating DNA end access; (3) if required, DNA end processing is carried on by non-core factors
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