XLF Regulates Filament Architecture of the XRCC4·Ligase IV Complex

Abstract

SUMMARY DNA ligase IV (LigIV) is critical for non-homologous end-joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in human cells, and LigIV activity is regulated by XRCC4 and XLF (XRCC4-like factor) interactions. Here, we employ X-ray scattering (SAXS) data to characterize three-dimensional arrangements in solution for full-length XRCC4, XRCC4 in complex with LigIV tandem BRCT domains and XLF, plus the XRCC4·XLF·BRCT2 complex. XRCC4 forms tetramers mediated through a head-to-head interface and the XRCC4 C-terminal coiled-coil region folds back on itself to support this interaction. The interaction between XLF and XRCC4 is also mediated via head-to-head interactions. In the XLF·XRCC4·BRCT complex, alternating repeating units of XLF and XRCC4·BRCT place the BRCT domain on one side of the filament. Collective results identify XRCC4 and XLF filaments suitable to align DNA molecules and function to facilitate LigIV end joining required for DSB repair in vivo.

Structural characterization of filaments formed by human Xrcc4–Cernunnos/XLF complex involved in nonhomologous DNA end-joining

“…This property arises from the distal positions of the interaction sites on both protein head domains and from the homodimeric nature of both X4 and Cernunnos. This observation agrees with low-resolution models obtained by SAXS using X4 1-140 and Cernunnos 1-248 constructs (30). In this study, the authors obtained the best fit to their experimental data by mixing tetramer, hexamer, and octamer assemblies of X4-Cernunnos.…”

“…In particular, single mutations at any of three Cernunnos residues (R64, L65, and L115) disrupt its interaction with X4 and abrogate Cernunnos function in several DNA repair assays, despite correct Cernunnos expression and localization (29). Low-resolution models of an X4-Cernunnos complex obtained by small angle X-ray scattering (SAXS) were recently reported (30). The results of these experiments supported a head-to-head interaction model between X4 and Cernunnos and predicted a mixture of oligomers, from tetramers to octamers.…”

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

“…have shown that XLF and XRCC4 form extended filaments (26,27,(38)(39)(40)(41)(42). Importantly, our SR imaging revealed that XRCC4 and XLF filaments exist in vivo, and also that LigIV appears to form filamentous structures in cells (Fig.…”

“…Moreover, the involvement of LigIV with these structures was uncertain (26). Our discovery that filaments do indeed form in cells, and that these localize to DSB sites, sheds new light on the physical organization of the NHEJ repair complex (Fig.…”

“…Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15)(16)(17)(18); however, recent experiments indicate that DNAPKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered DSBs (19)(20)(21)(22)(23)(24)(25). In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26)(27)(28). Whether such filaments mediate repair in vivo has not yet been determined.…”