XRCC4 and XLF form long helical protein filaments suitable for DNA end protection and alignment to facilitate DNA double strand break repair

Abstract

DNA double strand breaks (DSBs), induced by ionizing radiation (IR) and endogenous stress including replication failure, are the most cytotoxic form of DNA damage. In human cells, most IR-induced DSBs are repaired by the nonhomologous end joining (NHEJ) pathway. One of the most critical steps in NHEJ is ligation of DNA ends by DNA ligase IV (LIG4), which interacts with, and is stabilized by, the scaffolding protein X-ray cross-complementing gene 4 (XRCC4). XRCC4 also interacts with XRCC4-like factor (XLF, also called Cernunnos); yet, XLF has been one of the least mechanistically understood proteins and precisely how XLF functions in NHEJ has been enigmatic. Here, we examine current combined structural and mutational findings that uncover integrated functions of XRCC4 and XLF and reveal their interactions to form long, helical protein filaments suitable to protect and align DSB ends. XLF-XRCC4 provides a global structural scaffold for ligating DSBs without requiring long DNA ends, thus ensuring accurate and efficient ligation and repair. The assembly of these XRCC4-XLF filaments, providing both DNA end protection and alignment, may commit cells to NHEJ with general biological implications for NHEJ and DSB repair processes and their links to cancer predispositions and interventions.

Figure 1. A model for non-homologous end joining

NHEJ is proposed to occur in three stages, (i) end detection and tethering, (ii) end processing to remove non-ligatable end groups and (iii) ligation of the DNA ends. In the first step, the DSBs are detected by the Ku70/80 heterodimer. Ku is required for recruitment of DNA-PKcs, which displaces Ku form the DNA end (Yoo and Dynan, 1999) and promotes DNA end-tethering and formation of a synaptic complex (DeFazio et al., 2002). Autophosphorylation of DNA-PKcs induces conformational changes (Hammel et al., 2010b) that may induce its dissociation from DNA ends (Dobbs et al., 2010) and regulate subsequent processing of DNA ends (reviewed in (Meek et al., 2008)) but when this occurs relative to end processing and recruitment of the XRCC4-Lig4 complex is not clear. Processing of the DNA ends is thought to involve Artemis (which interacts with DNA-PKcs (Ma et al., 2002)), PNKP (which interacts with CK2 phosphorylated XRCC4 (Koch et al., 2004)) and DNA polymerases of the pol X family (Nick McElhinny and Ramsden, 2004), however, precisely when each protein is recruited to and subsequently released from the NHEJ repair complex is not known (indicated by dashed lines and question marks). In the final step, the processed DNA ends are ligated by the LIG4-XRCC4 complex. XRCC4 and XLF may promote DNA end ligation by regulating the catalytic activity of LIG4 and by the formation of end bridging filaments, as described in the text. In the figure, Ku80 is shown in orange and Ku70 in red (orientation relative to DSB ends from (Walker et al., 2001)), DNA-PKcs is in blue, Artemis (Art) in grey, dimeric XRCC4 in green, dimeric XLF in purple, LIG4 in black and PNKP in pink.

Figure 2. Structures of XRCC4 and XLF

A: Cartoon showing the major regions of XRCC4. Boundaries between the head, stalk and C-terminal (CTR) domains are from (Junop et al., 2000; Sibanda et al., 2001). Phosphorylation sites (shown in red) are taken from Phosphosites.org (search term http://www.phosphosite.org/proteinAction.do?id=18166&showAllSites=true). B: Cartoon showing major regions of XLF. Boundaries between head (orange) and stalk (grey) domains are from (Andres et al., 2007; Li et al., 2008). Phosphorylation sites (shown in red) are from (Yu et al., 2008). C: Crystal structure of XRCC4 with positions of head (orange) and stalk (green) domains from (Junop et al., 2000; Sibanda et al., 2001) and the predicted position of the unstructured CTR (light-magenta) from SAXS (Hammel et al., 2011; Hammel et al., 2010b). Phosphorylation sites are in red, as in panel A. D: Crystal structure of XLF with predicted position of XLF-CTR (light-magenta) from SAXS (Hammel et al., 2011; Hammel et al., 2010b). Phosphorylation sites are in red, as above. The extreme CTR of XLF, shown to interact with Ku (Yano et al., 2011) and the region shown to support filament formation (Hammel et al., 2011) are indicated.

Figure 3. The XRCC4-XLF interface

Top: Structures of XRCC4 homodimers (left) and XLF homodimers (right) coloured as in Figure 2, showing the location of XLF-L115 in the XRCC4-XLF interface. Middle: Structure above, rotated by 90°. Lower: Close-up of XRCC4-XLF interface showing locations of E55, M59, M6, F106, R107 and L108 for XRCC4 (green) and R64, R65 and L65 (blue). The formation of a beta-zipper structure upon complexation is highlighted (see (Hammel et al., 2011) for details). The head domains of XRCC4 and XLF are shown in gold as above.

Figure 4. Models for XRCC4-XLF filaments in NHEJ

A: Model of filaments composed of XRCC4 (blue) and XLF (magenta). Two parallel filaments are shown as observed in the crystal and in solution (Hammel et al., 2011). On the right, the parallel filaments are rotated by 90° to show the central channel or pore (see text for details). B: Model of XRCC4-XLF super-helical bundles, coloured as in panel A, with dsDNA (green) wrapping around the filament as proposed by (Hammel et al., 2011) (top) or within the filament as proposed by (Andres et al., 2012) (bottom). C: Possible positions of the Ku heterodimer (grey) and DNA-PKcs (peach) relative to DNA-protein filaments (adapted from (Hammel et al., 2011)). D: Possible models for the position of XRCC4-XLF filaments relative to the DSB: In all panels, XRCC4 is shown in blue, XLF in magenta, Ku in grey and dsDNA in green as in panels A–C. The positions of the DSB ends are marked by triangles. Possible access points for DNA-PKcs, processing enzymes and/or LIG4 are indicated by the arrows. In all models, it is possible that filament dynamics and interactions with other proteins are regulated by protein phosphorylation, possibly of the CTRs of XRCC4 and XLF. We speculate that filaments promote synapsis of DNA ends and protect ends from nuclease degradation, which may affect pathway choice as well as DNA fidelity, see text for details. (i) XRCC4/XLF filaments form proximal to the DSB, supporting and aligning dsDNA either side of the DSB. How DNA-PKcs, processing enzymes (for example PNKP) and LIG4 gain access to the DNA ends is presently unknown. One possibility is that XRCC4-XLF filaments form only after DNA-PKcs autophosphorylation and release (see Fig. 1). Also, since cells contain excess of XRCC4 over LIG4, it is possible that XRCC4 in filaments is devoid of LIG4 and that ligation is carried out by a soluble pool of XRCC4-LIG4, perhaps in complex with PNKP. It has been suggested that the presence of LIG4 near the DNA ends may disrupt the XRCC4/XLF filaments, thereby providing a possible mechanism to both terminate filament formation and provide LIG4 with access to the DNA ends. Protein phosphorylation may also play a role in remodeling the filament ends (see text for details). (ii) The Ku-DNA-PKcs complex forms at the DSB ends and the XRCC4-XLF filament forms distal to the break. This model is attractive in that the DNA ends would be accessible for binding by DNA-PKcs. Subsequent DNA-PKcs autophosphorylation and dissociation/remodeling might then allow processing enzymes such as PNKP and Artemis to gain access to the DSB ends (see Fig. 1). This model would also be consistent with two pools of XRCC4, one in filamentous form and one that interacts with LIG4. (iii) Filaments perform a bridging role, spanning two Ku-DNA complexes. Remodeling of filament ends, possibly by protein phosphorylation, could allow access of processing enzymes and LIG4 to DNA termini. (iv) Given the dimensions of the internal channel, another possibility is that the filaments could form around two dsDNA molecules, allowing processing and ligation to occur at the exposed DNA termini.