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

Abstract

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4 1-157 -Cernunnos 1-224 complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4 1-157 and Cernunnos 1-224 homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu 55 , Asp 58 , Met 61 , and Phe 106 ) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.D NA double-strand breaks (DSBs) are the most toxic DNA lesions in the genome, and unrepaired DSBs can cause large-scale losses of genetic information through chromosome rearrangement (1, 2). These DNA damages result from exposure to exogenous damaging agents, such as ionizing radiation, radiomimetic compounds, and topoisomerase inhibitors. DSBs are also obligate intermediates in several recombination processes in vertebrates, including antigen receptor gene rearrangement, V(D)J recombination (3, 4). In higher eukaryotes, DSBs are repaired by several mechanisms, among which nonhomologous end-joining (NHEJ) represents the major pathway, particularly when sister chromatids are not available (5). Deficiency in the NHEJ machinery results in sensitivity to ionizing radiation and severe combined immune deficiencies in humans and mice due to abortive V(D)J recombination (6). NHEJ is orchestrated by at least seven proteins. The Ku70/Ku80 heterodimer adopts a preformed ring-shaped structure that recognizes and encircles the duplex DNA ends at the DSB (7). Ku70/Ku80 recruits a 469-kDa serine/threonine protein kinase, the DNA-PK catalytic subunit (DNA-PKcs), via a direct interaction and shifts about 10 bp inward so that DNA-PKcs acquires a position at the terminus through its large open-ring cradle structure (8). Upon association with Ku and DNA, DNA-PKcs is activated and phosphorylates several proteins including itself and Ku70/Ku80. The DNA-PK holoenzyme, constituted by Ku and DNA-PKcs, plays a central role in NHEJ. Among other functions, DNA-PK mediates the end-bridging of the DSB extremities (9), regulates access to the DNA ends by processing enzymes such as the DNA-PKcs-associated Artemis nuclease (10, 11), and recruits the Xrcc4-ligase IV complex to DNA ends for the ligation step (12). The Xrcc4-ligase IV complex carries out the final joinin...

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

“…previous reports that XLF/XRCC4 can form filaments in the absence of DNA (41). When the proteins Ku, LX, and XLF were reacted, we observed similar filaments when staining for LigIV ( Fig.…”

“…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.…”

“…The presence of filaments at either side of the break is advantageous, allowing for more pairing configurations compared with pairing events occurring only at the ends, thereby increasing the probability of pairing. When two Caterpillar structures meet, the XRCC4/XLF/LigIV filaments pair in either a direct end-to-end or an adjacent configuration, forming Butterfly structures (41,42).…”

Cernunnos Deficiency Reduces Thymocyte Life Span and Alters the T Cell Repertoire in Mice and Humans

“…Accordingly, Cernu Ϫ/Ϫ mice crossed onto the P53 KO background did not develop B cell lymphoma, as opposed to the case with other NHEJ deficiency situations (26). We propose a speculative yet plausible explanation for this intriguing observation, based on our recent report on the Cernunnos-Xrcc4 complex structure (27). Crystals of Cernunnos-Xrcc4 complexes revealed that both homodimers associate with each other in long filaments through their head domains, helping to tether the broken DNA ends by creating a "DNA ligation synapse" (27)(28)(29)(30).…”

XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination

“…Since its discovery in 2005, several functions have been described for XLF. It stimulates LIG4 activity, 21,22 and it can form long filaments with XRCC4 that keep the DNA ends together in a ligation synapse, 24,[48][49][50][51] and XLF is essential for gap-filling by polymerase (pol) l and polm during NHEJ. The latter function is interesting, as poll and polm belong to the same polX family of polymerases as For personal use only.…”