| 2006 |
XLF (Cernunnos/NHEJ1) directly interacts with the XRCC4-DNA Ligase IV complex in vitro and in vivo, and siRNA-mediated knockdown of XLF causes radiosensitivity and impaired NHEJ in human cell lines. Re-introduction of wild-type XLF into XLF-deficient 2BN cells corrects radiosensitivity and NHEJ defects, establishing XLF as a core component of the mammalian NHEJ apparatus. |
In vitro pulldown, Co-IP, siRNA knockdown, complementation assay, NHEJ reporter assay |
Cell |
High |
16439204 16439205
|
| 2006 |
Cernunnos/XLF physically interacts with the XRCC4–DNA Ligase IV complex and is the homolog of the yeast NHEJ factor Nej1p, placing it within the evolutionarily conserved ligation module of NHEJ. |
Co-immunoprecipitation, sequence/structural homology analysis, yeast two-hybrid |
The Journal of biological chemistry |
High |
16571728
|
| 2006 |
XLF family proteins (including S. pombe ortholog) bind DNA directly and interact with the Ligase IV–XRCC4 complex to promote DSB ligation, demonstrating evolutionary conservation of this enzymatic core. |
DNA-binding assay, co-precipitation, NHEJ ligation assay in S. pombe and human cells |
The Journal of biological chemistry |
Medium |
17038309
|
| 2007 |
Cernunnos/XLF stimulates XRCC4/DNA Ligase IV-mediated ligation of mismatched and noncohesive DNA ends 8- to 150-fold depending on mismatch type; it also promotes ligation of a 3′ overhang hydroxyl to the 5′ phosphate of a blunt end, providing a mechanism for 3′ overhang retention during V(D)J recombination. |
In vitro NHEJ ligation assay with purified proteins (Ku, DNA-PKcs, XRCC4/LigIV, Cernunnos) |
Proceedings of the National Academy of Sciences of the United States of America |
High |
17470781
|
| 2007 |
XLF binds DNA in a length-dependent manner consistent with C-terminal α-helices orienting parallel to the DNA helix, directly interacts with purified XRCC4–DNA Ligase IV complex, and stimulates its ligation activity. A patient-derived XLF R57G mutant retains stimulatory activity in vitro but fails to translocate to the nucleus, identifying nuclear import as the basis for the NHEJ defect in that patient. |
In vitro ligation assay with purified proteins, DNA-binding assay, nuclear localization assay with mutant protein |
The Journal of biological chemistry |
High |
17317666
|
| 2007 |
Ku is essential for XLF recruitment to DSBs (live-cell laser micro-irradiation imaging); Ku–XLF interaction occurs on DNA and Ku stimulates XLF DNA binding. XRCC4 is dispensable for XLF recruitment but stabilizes XLF at DSBs (FRAP/photobleaching analysis). |
Live-cell imaging with laser micro-irradiation, FRAP, biochemical DNA-binding assay |
EMBO reports |
High |
18064046
|
| 2007 |
Crystal structure of human XLF (residues 1–224) reveals a homodimeric protein with structural homology to XRCC4 but with a compact, folded helical C-terminal region (two turns and a twist) rather than XRCC4's extended coiled-coil; mutational analysis of XLF and XRCC4 identified a potential head-domain interaction interface. |
X-ray crystallography, mutagenesis |
Molecular cell |
High |
18158905
|
| 2007 |
Crystal structure of XLF (1–233) homodimer at 2.3 Å confirms structural similarity to XRCC4 but shows a shorter, reversed coiled-coil giving a four-helical bundle. SPR demonstrates XLF–XRCC4 dimer interactions, most consistent with head-to-head contacts in a 2:2:1 XRCC4:XLF:Ligase IV complex. |
X-ray crystallography, size-exclusion chromatography, analytical ultracentrifugation, SAXS, surface plasmon resonance |
The EMBO journal |
High |
18046455
|
| 2007 |
XRCC4 and XLF (Nej1/Lif1 in yeast) form stable coiled-coil homodimers rather than heterodimers; XLF–XRCC4 interactions are mediated through the globular head of XRCC4/Lif1 contacting N- and C-terminal domains of XLF/Nej1 (different regions for XLF vs Nej1), with additional direct XLF/Nej1–Ligase IV contacts distinct from the stable Lif1–Ligase IV coiled-coil interaction. |
Yeast two-hybrid, co-precipitation, domain deletion analysis |
DNA repair |
Medium |
17567543
|
| 2007 |
XLF/Cernunnos stimulates ligation of both incompatible and compatible DNA ends by XRCC4–DNA Ligase IV at physiological Mg2+; at high Mg2+ it stimulates only incompatible ends, suggesting charge-neutralization between DNA ends within the ligase complex. XRCC4–DNA Ligase IV also ligates poly-dT single-stranded DNA and long dT overhangs independently of Ku and XLF. |
In vitro ligation assay with purified recombinant proteins |
Nucleic acids research |
High |
17717001
|
| 2007 |
In living cells, XLF and XRCC4 are independently recruited to Ku-bound DSBs rather than sequentially; XRCC4 modulates the exchange rate of XLF at DSBs, and DNA-PKcs stabilizes XRCC4 at DSBs (two-phase model of NHEJ assembly). |
Live-cell imaging, laser micro-irradiation, FRAP |
Cell cycle (Georgetown, Tex.) |
Medium |
18418068
|
| 2007 |
In XLF/Cernunnos-deficient human cell extracts, gap filling by DNA polymerases λ and μ on aligned DSB ends is completely absent; addition of recombinant XLF restores both gap filling and end joining of partially complementary overhangs, and immunodepletion of polymerase λ eliminates XLF-dependent gap filling, identifying XLF as essential for polymerase activity during NHEJ. |
Cell-free NHEJ assay with whole-cell extracts, immunodepletion, recombinant protein complementation, dideoxynucleotide trapping of intermediates |
Nucleic acids research |
High |
19420065
|
| 2007 |
Cernunnos-XLF is co-recruited with core NHEJ components to DSB-damaged chromatin and is phosphorylated by DNA-PKcs in cells. DNA Ligase IV (not XRCC4) is required for Cernunnos association with the XRCC4/Ligase IV complex and for its mobilization to damaged chromatin; conversely, XLF deficiency does not affect XRCC4/Ligase IV association or their recruitment to DSBs. |
Detergent-based chromatin fractionation, Co-IP, immunoblot |
The Journal of biological chemistry |
Medium |
17720816
|
| 2008 |
XLF promotes re-adenylation of the DNA Ligase IV–XRCC4 complex after ligation (in situ recharging), enabling a single complex to complete double-stranded ligation. XLF also enhances end-bridging in an ATP-independent manner. XLF is a weakly bound partner of the tight Ligase IV–XRCC4 complex and is dispensable for Ligase IV–XRCC4 stability. |
Biochemical adenylation assay, ligation assay, co-precipitation, cellular DSB repair assay |
Nucleic acids research |
High |
19056826
|
| 2008 |
DNA-PK phosphorylates XLF at serines 245 and 251 in vitro and in vivo; Ser245 is phosphorylated by DNA-PK and Ser251 by ATM in vivo. However, phospho-blocking alanine mutations at these sites do not affect XLF–DNA interaction, recruitment to laser-induced DSBs, or ability to complement DSB repair in XLF-deficient cells, indicating these phosphorylations are not required for NHEJ. |
In vitro kinase assay, mass spectrometry, site-directed mutagenesis, live-cell imaging, complementation assay |
DNA repair |
High |
18644470
|
| 2008 |
In adenovirus-infected cells, loss of DNA Ligase IV (via viral E1B 55k/E4 34k-mediated degradation) results in loss of DNA-binding activity by both XRCC4 and XLF, suggesting that Ligase IV is required for the intrinsic DNA-binding activities of XRCC4 and XLF. |
Adenovirus infection, immunoblot, DNA-binding assay, ligase IV–deficient cell lines |
Nucleic acids research |
Medium |
18782835
|
| 2010 |
Combined deficiency of XLF and ATM nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. XLF and ATM have functionally redundant roles in NHEJ mediated by ATM kinase activity; H2AX inactivation in XLF-deficient pro-B cells also causes V(D)J recombination defects with degradation of unjoined ends, revealing an end-protection role for H2AX. |
Mouse genetics (double knockout), V(D)J recombination assay, chromosomal break analysis, flow cytometry |
Nature |
High |
21160472
|
| 2010 |
Systematic mutagenesis identified three XLF residues (Arg64, Leu65, Leu115) in the globular head domain essential for interaction with XRCC4 and for XLF function in DNA repair; structural docking validated this interaction surface. |
Site-directed mutagenesis, co-immunoprecipitation, DNA repair assay, structural modeling |
The Journal of biological chemistry |
High |
20558749
|
| 2010 |
SAXS analysis reveals that XLF and XRCC4 interact via head-to-head interfaces to form extended filaments in solution; in the XLF·XRCC4·BRCT complex, alternating repeating units place the BRCT domain on one side of the filament, suggesting a scaffold for aligning DNA molecules during LigIV-mediated end joining. |
Small-angle X-ray scattering (SAXS) |
Structure (London, England : 1993) |
Medium |
21070942
|
| 2011 |
Crystal structure (5.5 Å) of the XRCC4(1–157)–Cernunnos(1–224) complex reveals a filament arrangement of alternating homodimers mediated by repeated head-domain interactions. Structure-based mutagenesis and calorimetry identified four XRCC4 residues (Glu55, Asp58, Met61, Phe106) essential for Cernunnos interaction. |
X-ray crystallography, transmission electron microscopy, structure-based site-directed mutagenesis, isothermal titration calorimetry |
Proceedings of the National Academy of Sciences of the United States of America |
High |
21768349
|
| 2011 |
Crystal structure of the XLF–XRCC4 complex combined with SAXS and mutational analysis shows alternating XLF and XRCC4 head domains forming parallel super-helical filaments. XLF Leu-115 ('Leu-lock') inserts into a hydrophobic pocket on XRCC4 (Met-59, Met-61, Lys-65, Lys-99, Phe-106, Leu-108); the positively charged channel of the filament binds DNA and aligns ends for ligation. |
X-ray crystallography, SAXS, mutagenesis, biochemical assays |
The Journal of biological chemistry |
High |
21775435
|
| 2011 |
XLF interacts with Ku via its C-terminal region; a small C-terminal deletion of XLF abolishes both DSB recruitment and Ku–XLF interaction, and also markedly reduces XLF–XRCC4 interaction even though the XRCC4-binding site on the N-terminal domain remains intact, demonstrating that Ku–XLF interaction is essential for molecular assembly of NHEJ factors. |
Domain deletion analysis, live-cell imaging (laser micro-irradiation), Co-IP |
FEBS letters |
Medium |
21349273
|
| 2012 |
XRCC4 and XLF complexes bridge DNA molecules in a DNA Ligase IV-independent manner (DNA-bridging and -binding assays); mutational analysis of C-terminal tails identifies specialized functions in complex formation, DNA interaction, and DNA Ligase IV interaction. Crystal structure of extended XLF–XRCC4 filament at 3.94 Å supports a bridging role. |
DNA-bridging assay, DNA-binding assay, electron microscopy, X-ray crystallography, mutagenesis |
Nucleic acids research |
High |
22287571
|
| 2012 |
Ablating XRCC4's affinity for XLF results in a deficit in V(D)J coding end joining but not signal end joining in cells, suggesting XRCC4/XLF complexes hold DNA ends together in a manner stringently required for coding ends but dispensable for signal ends. DNA-PK phosphorylation of XRCC4/XLF complexes disrupts DNA bridging in vitro. |
Structure-based mutagenesis, V(D)J recombination assay, DNA-bridging assay, kinase assay |
Nucleic acids research |
High |
22228831
|
| 2015 |
Akt phosphorylates XLF at Thr181, triggering its dissociation from the DNA Ligase IV/XRCC4 complex and promoting interaction with 14-3-3β, which leads to XLF cytoplasmic retention and subsequent SCF(β-TRCP)-mediated degradation. A cancer-patient-derived XLF-R178Q mutant that is deficient in Thr181 phosphorylation shows elevated DNA damage tolerance. |
In vitro kinase assay, Co-IP, cellular fractionation, ubiquitination assay, mutagenesis |
Molecular cell |
High |
25661488
|
| 2015 |
PAXX interacts directly with Ku (not XLF or XRCC4) and is recruited to DNA damage sites. PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core NHEJ factors on damaged chromatin; combined depletion of PAXX and XLF is more severely defective in DSB repair than either single deficiency. |
Crystal structure, Co-IP, CRISPR-Cas9 knockout, in vitro ligation assay, chromatin fractionation |
Science (New York, N.Y.) |
High |
25574025
|
| 2016 |
Using optical tweezers with fluorescence microscopy, XLF stimulates the binding of XRCC4 to DNA; XRCC4–XLF heteromeric complexes diffuse rapidly along DNA (sliding sleeves) and robustly bridge two independent DNA molecules with mobile, sleeve-like structures, suggesting they can rapidly reconnect broken ends and hold them together. |
Dual/quadruple-trap optical tweezers, single-molecule fluorescence microscopy |
Nature |
High |
27437582
|
| 2018 |
Crystal structures of the XLF Ku-binding motif (X-KBM) bound to a Ku–DNA complex show the X-KBM occupying an internal pocket of the Ku80 α/β domain formed by an unprecedented large outward rotation of that domain. Mutations disrupting the X-KBM binding site on Ku80 compromise both efficiency and accuracy of end joining and increase cellular radiosensitivity. |
X-ray crystallography, mutagenesis, laser irradiation recruitment assay, end-joining assay, radiosensitivity assay |
Nature structural & molecular biology |
High |
30291363
|
| 2018 |
Single-molecule fluorescence imaging in Xenopus egg extract shows that a single XLF dimer (not a filament) binds DNA substrates just before formation of a ligation-competent synaptic complex. Interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient synaptic complex formation. |
Single-molecule fluorescence imaging, Xenopus egg extract NHEJ assay, mutagenesis |
Nature structural & molecular biology |
High |
30177755
|
| 2017 |
Phospho-mimicking mutations at fourteen DNA-PK/ATM phosphorylation sites in the C-terminal tails of both XRCC4 and XLF concomitantly impair stability and DNA-bridging capacity of XRCC4/XLF complexes without affecting their ability to stimulate LIG4 activity, indicating that phosphorylation regulates DNA bridging but not ligase stimulation. |
Site-directed mutagenesis, DNA-bridging assay, LIG4 stimulation assay |
eLife |
High |
28500754
|
| 2017 |
PAXX, through its interaction with Ku70 (forming a stable ternary complex with Ku–DNA), provides weak stimulation of LIG4/XRCC4 activity that is unmasked only by XLF ablation, demonstrating that PAXX is an accessory c-NHEJ factor with functions largely overlapping XLF. |
In vitro ligation assay, Co-IP, PAXX-deficient cell analysis, shRNA knockdown |
Cell reports |
Medium |
27705800
|
| 2017 |
PAXX promotes KU accumulation at DSBs (measured by quantitative ChIP/imaging), while XLF enhances LIG4 recruitment to breaks without affecting KU dynamics, demonstrating distinct and complementary molecular functions at DNA ends in vivo. |
Mouse genetics (double knockout), quantitative chromatin immunoprecipitation, immunofluorescence at DSBs |
Nature communications |
High |
28051062
|
| 2018 |
For end joining without indels, XLF requires synergistic function of two distinct binding domains: L115 (XRCC4-binding) and C-terminal lysines (KU/DNA-binding); disruption of one sensitizes XLF to mutations at the dimer interface, revealing interdependent functional architecture. |
Chromosomal EJ reporter assay (Cas9-induced breaks), site-directed mutagenesis, molecular dynamics simulation |
Nature communications |
Medium |
29950655
|
| 2018 |
PTEN promotes NHEJ by directly inducing expression of XLF (NHEJ1) through occupancy of the NHEJ1 gene promoter and recruitment of histone acetyltransferases PCAF and CBP; this activity is independent of PTEN phosphatase activity but requires K128 (a regulatory acetylation site on PTEN). |
Chromatin immunoprecipitation, co-immunoprecipitation, reporter assay, mutagenesis, NHEJ reporter assay |
Molecular cancer research : MCR |
Medium |
29739874
|
| 2019 |
XLF associates with the replication factor C (RFC) complex (a critical replisome component) and is found at replication forks; XLF undergoes CDC7-dependent phosphorylation, and XLF deficiency causes defects in replication fork progression and increased fork reversal. |
Co-immunoprecipitation, iPOND (replication fork isolation), CDC7 kinase assay, DNA fiber assay |
The Journal of cell biology |
Medium |
31123184
|
| 2020 |
Single-molecule FRET in Xenopus egg extract shows that the intrinsically disordered C-terminal tail of XLF, together with its Ku-binding motif (KBM) at the extreme C-terminus, is required for close DNA end alignment during synapsis. A minimal tail length (but not specific sequence) is necessary; the tail tethers XLF to Ku while allowing XRCC4 interactions that enable synaptic complex formation. |
Single-molecule FRET, Xenopus egg extract NHEJ assay, mutagenesis (tail truncation/scrambling) |
eLife |
High |
33289484
|
| 2023 |
Cryo-EM structures show PAXX C-terminal KBM bound to Ku70/80, and PAXX bound to two alternate DNA-PK end-bridging dimers mediated by either Ku80 or XLF. PAXX and XLF can simultaneously bind the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers; residues critical for Ku70/PAXX interaction were identified and validated in vitro and in cells. |
Cryo-EM, X-ray crystallography, mutagenesis, in vitro binding assay, cellular end-joining assay |
Science advances |
High |
37256950
|
| 2024 |
The C-terminal regions (CTRs) of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions; these CTR interactions promote robust cellular NHEJ activity and drive formation of XLF and X4L4 condensates in vitro that can recruit effectors and critically stimulate DNA end ligation. |
NMR (solution-state), biochemical assays, condensate formation assay, mutagenesis |
Nature structural & molecular biology |
High |
38898102
|
| 2025 |
XLF is lactylated at K288 within its Ku-binding motif (X-KBM) by the acetyltransferase GCN5 in a process triggered by DNA damage–induced ATM-mediated GCN5 phosphorylation. Lactylation of K288 enhances XLF–Ku80 binding and XLF recruitment to DSBs, increasing NHEJ efficiency; cryo-EM shows lactylated X-KBM forms a more extensive interface with Ku70/80 inducing Ku80 vWA domain conformational changes. |
Cryo-EM, in vitro lactylation assay, Co-IP, ATM/GCN5 kinase assay, mutagenesis, NHEJ reporter assay |
Molecular cell |
High |
40680721
|