Evolutionary and Functional Conservation of the DNA Non-homologous End-joining Protein, XLF/Cernunnos

Abstract

Non-homologous end-joining is a major pathway of DNA double-strand break repair in mammalian cells, deficiency in which confers radiosensitivity and immune deficiency at the whole organism level. A core protein complex comprising the Ku70/80 heterodimer together with a complex between DNA ligase IV and XRCC4 is conserved throughout eukaryotes and assembles at double-strand breaks to mediate ligation of broken DNA ends. In Saccharomyces cerevisiae an additional NHEJ protein, Nej1p, physically interacts with the ligase IV complex and is required in vivo for ligation of DNA double-strand breaks. Recent studies with cells derived from radiosensitive and immune-deficient patients have identified the human protein, XLF (also named Cernunnos), as a crucial NHEJ protein. Here we show that XLF and Nej1p are members of the same protein superfamily and that this family has members in diverse eukaryotes. Indeed, we show that a member of this family encoded by a previously uncharacterized open-reading frame in the Schizosaccharomyces pombe genome is required for NHEJ in this organism. Furthermore, our data reveal that XLF family proteins can bind to DNA and directly interact with the ligase IV-XRCC4 complex to promote DSB ligation. We therefore conclude that XLF family proteins interact with the ligase IV-XRCC4 complex to constitute the evolutionarily conserved enzymatic core of the NHEJ machinery.The repair of DNA double-strand breaks (DSBs) 3 is crucial for the maintenance of genomic integrity (1, 2). In mammalian cells, non-homologous end-joining (NHEJ) is the predominant pathway for DSB repair, particularly in the G 0 and G 1 phases of the cell cycle, and is mediated by the Ku70/Ku80 heterodimer, the DNA-dependent protein kinase catalytic subunit (DNAPKcs), XRCC4 and DNA ligase IV (3, 4). It is believed that during NHEJ, newly created DSB ends are bound by the Ku heterodimer (5, 6) and subsequently, DNA-PKcs is recruited upon inward translocation of Ku70/Ku80 (7-9). The Ku-DNA-PKcs complex is thought to stabilize the DNA ends and prevent extensive end resection (10), and most likely also plays an important role in aligning the DNA termini (11-14). The DNAPKcs-Ku complex then promotes the recruitment of a preformed complex between XRCC4 and DNA ligase IV (ligase IV), which mediates the final ligation steps of the NHEJ process (8, 15).XRCC4 is a ϳ36-kDa protein that consists of a globular N-terminal domain followed by a protruding coiled-coil arm (16,17). It has been well established that XRCC4 forms homodimers, as well as higher order multimers (16 -19), and that an active ligase IV complex is most likely composed of one XRCC4 homodimer bound to one ligase IV molecule (17, 18). Significantly, XRCC4 not only promotes ligase IV activity but is also required for ligase protein stability (20,21). In addition to the above core set of NHEJ proteins, a number of other factors have been identified that are required for the efficient repair of DSBs by NHEJ. These factors, most prominently Artemis (22, 23), polynucleotide kinase ...

V(D)J and immunoglobulin class switch recombinations: a paradigm to study the regulation of DNA end-joining

“…Cernunnos orthologues (referenced as Nej1p or XLF1) have further been found in many eukaryotes demonstrating that Nej1p and Cernunnos/XLF belong to the same protein family (Callebaut et al, 2006;Hentges et al, 2006;Cavero et al, 2007). Nej1p in yeast interacts with the XRCC4 orthologue Lif1p, suggesting that Nej1p and Cernunnos/XLF have conserved an analogous function throughout evolution.…”

“…Cernunnos is ubiquitously expressed and localized predominantly in the nucleus. Sequence analysis revealed that Cernunnos shares structural features with XRCC4 revealing the existence of a new protein family (Callebaut et al, 2002;Ahnesorg et al, 2006;Hentges et al, 2006). Based on the XRCC4 structure (Junop et al, 2000;Sibanda et al, 2001), one can predict a similar conformation for Cernunnos, that is a globular head domain followed by a coil-coiled tail Callebaut et al, 2006;Hentges et al, 2006).…”

“…Sequence analysis revealed that Cernunnos shares structural features with XRCC4 revealing the existence of a new protein family (Callebaut et al, 2002;Ahnesorg et al, 2006;Hentges et al, 2006). Based on the XRCC4 structure (Junop et al, 2000;Sibanda et al, 2001), one can predict a similar conformation for Cernunnos, that is a globular head domain followed by a coil-coiled tail Callebaut et al, 2006;Hentges et al, 2006). Cernunnos/XLF, like XRCC4, can bind DNA in a sequence-independent manner (Hentges et al, 2006;Lu et al, 2007 Deshpande and Wilson, 2007).…”

“…Based on the XRCC4 structure (Junop et al, 2000;Sibanda et al, 2001), one can predict a similar conformation for Cernunnos, that is a globular head domain followed by a coil-coiled tail Callebaut et al, 2006;Hentges et al, 2006). Cernunnos/XLF, like XRCC4, can bind DNA in a sequence-independent manner (Hentges et al, 2006;Lu et al, 2007 Deshpande and Wilson, 2007). Their globular head domains could drive their direct association.…”

“…Their globular head domains could drive their direct association. Both Cernunnos/XLF and XRCC4 appear to directly interact with DNA-LigaseIV but the Cernunnos/XLF-DNA-LigaseIV interaction is very weak (Hentges et al, 2006;Deshpande and Wilson, 2007). The exact nature of the complex(es) formed between XRCC4, DNA-LigaseIV and Cernunnos/XLF remains to be clearly established, but one can anticipate that differential complex formation may have important regulatory function for the DNA-end ligation reaction during the NHEJ process.…”