Weak Sequence Homology Research Articles

Synthetic self-adjuvanted multivalent Mucin 1 (MUC1) glycopeptide vaccines with improved in vivo antitumor efficacy.

The tumor-associated glycoprotein Mucin 1 (MUC1) is aberrantly glycosylated on cancer cells and is considered a promising target for antitumor vaccines. The weak immunogenicity and low sequence homology of mouse mucins and human MUC1 are the main obstacles for the development of vaccines. Herein, a self-adjuvanted strategy combining toll-like receptor 2 lipopeptide ligands and T-cell epitopes and the multivalent effect were used to amplify the immune response and evade the unpredictable immunogenicity, generating two self-adjuvanted three-component MUC1 vaccines (mono- and trivalent MUC1 vaccines). To simulate the aberrantly glycosylated MUC1 glycoprotein, the MUC1 tandem repeat peptide was bounded with Tn antigens at T9, S15, and T16, and served as B-cell epitopes. Results showed that both vaccines elicited a robust antibody response in wild-type mice compared with a weaker response in MUC1 transgenic mice. The trivalent vaccine did not elevate the antibody response level compared with the monovalent vaccine; however, a more delayed tumor growth and prolonged survival time was realized in wild-type and transgenic mouse models treated with the trivalent vaccine. These results indicate that the self-adjuvanted three-component MUC1 vaccines, especially the trivalent vaccine, can trigger robust antitumor effects regardless of sequence homology, and, therefore, show promise for clinical translation.

The soluble domains of Gpi8 and Gaa1, two subunits of glycosylphosphatidylinositol transamidase (GPI-T), assemble into a complex

Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi823-306) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaa1. The complex between GST-Gpi823-306 (α) and His6-Gaa150-343 (β) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an α2β2 stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer.

The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.

The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box2,” which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.

Characterization of a Novel Orthomyxo-like Virus Causing Mass Die-Offs of Tilapia.

ABSTRACTTilapia are an important global food source due to their omnivorous diet, tolerance for high-density aquaculture, and relative disease resistance. Since 2009, tilapia aquaculture has been threatened by mass die-offs in farmed fish in Israel and Ecuador. Here we report evidence implicating a novel orthomyxo-like virus in these outbreaks. The tilapia lake virus (TiLV) has a 10-segment, negative-sense RNA genome. The largest segment, segment 1, contains an open reading frame with weak sequence homology to the influenza C virus PB1 subunit. The other nine segments showed no homology to other viruses but have conserved, complementary sequences at their 5′ and 3′ termini, consistent with the genome organization found in other orthomyxoviruses. In situ hybridization indicates TiLV replication and transcription at sites of pathology in the liver and central nervous system of tilapia with disease.

Production and characterisation of Epstein-Barr virus helicase-primase complex and its accessory protein BBLF2/3.

The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.

Protein structure prediction provides comparable performance to crystallographic structures in docking-based virtual screening

Structure based virtual screening has largely been limited to protein targets for which either an experimental structure is available or a strongly homologous template exists so that a high-resolution model can be constructed. The performance of state of the art protein structure predictions in virtual screening in systems where only weakly homologous templates are available is largely untested. Using the challenging DUD database of structural decoys, we show here that even using templates with only weak sequence homology (<30% sequence identity) structural models can be constructed by I-TASSER which achieve comparable enrichment rates to using the experimental bound crystal structure in the majority of the cases studied. For 65% of the targets, the I-TASSER models, which are constructed essentially in the apo conformations, reached 70% of the virtual screening performance of using the holo-crystal structures. A correlation was observed between the success of I-TASSER in modeling the global fold and local structures in the binding pockets of the proteins versus the relative success in virtual screening. The virtual screening performance can be further improved by the recognition of chemical features of the ligand compounds. These results suggest that the combination of structure-based docking and advanced protein structure modeling methods should be a valuable approach to the large-scale drug screening and discovery studies, especially for the proteins lacking crystallographic structures.

Structural basis for the recognition of Eps15 by the SGIP1 μ homology domain

Clathrin-mediated endocytosis (CME) is a process for eukaryotic cells to internalize extracellular molecules. FCHo1 and FCHo2 are involved in the initial clathrin assembly step of CME [1, 2]. These proteins contain the lipid-binding EFC/F-BAR domain at the N-terminus [3], and the μ homology domain (μHD) at the C-terminus. The μHDs of these proteins interact with a region rich in a repeated sequence motif, Asp-Pro-Phe (DPF), of an endocytic scaffold protein, Eps15. Eps15 contains fifteen DPF motifs. SGIP1 is also involved in CME and contains the μHD highly homologous to those of FCHo1/2 at the C-terminus, which also interacts with Eps15. The μHDs of these proteins share weak amino-acid sequence homology with the μ subunits of the adaptor protein complexes, such as AP-2, which links cargo proteins and clathrin in CME. To investigate the mechanism of Eps15 recognition by the FCHo1/FCHo2/SGIP1 μHDs, we first identified the minimal Eps15 fragment retaining the ability to interact with the μHD by the interaction studies using the SGIP1 μHD. We found that two Eps15-derived 11-residue peptides each containing two DPF motifs connected by a short 2–3 residue linker interact with the SGIP1 μHD with modest affinities (Kd = 15–20 μM). In contrast, peptides containing only one DPF motif did not bind to the μHD. Thus, the SGIP1 μHD requires two DPF motifs for binding. Moreover, the structures of the SGIP1 μHD in complex with the peptides containing two DPF motifs revealed that the SGIP1 μHD extensively recognizes the two adjacent tandem DPF motifs, while not contacting the flanking residues, consistent with the interaction studies. This mode of recognition is distinct from that of the Eps15 DPF motif recognition by the AP-2 appendage domains, providing the rationale of the recruitment of Eps15 by FCHo1/2 to the nascent endocytic sites, while allowing the FCHo1/2-bound Eps15 to recruit AP-2 complex with the different parts of the Eps15 DPF repeat region for clathrin assembly.

Structural and Biophysical Characterization of the Cytoplasmic Domains of Human BAP29 and BAP31

Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31.

Evolutionary mix-and-match with MFS transporters

Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ∼25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H(+) electrochemical gradient into a concentration gradient of substrate. MFS transporters are characterized by a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices. An alternating inverted triple-helix structural symmetry within the N- and C-terminal six-helix bundles suggests that the proteins arose by intragenic multiplication. However, despite similar features, MFS transporters share only weak sequence homology. Here, we show that rearrangement of the structural symmetry motifs in the Escherichia coli fucose permease (FucP) results in remarkable homology to lactose permease (LacY). The finding is supported by comparing the location of 34 point mutations in FucP to the location of mutants in LacY. Furthermore, in contrast to the conventional, linear sequence alignment, homologies between sugar- and H(+)-binding sites in the two proteins are observed. Thus, LacY and FucP likely evolved from primordial helix-triplets that formed functional transporters; however, the functional segments assembled in a different consecutive order. The idea suggests a simple, parsimonious chain of events that may have led to the enormous sequence diversity within the MFS.

Structures of bacterial biosynthetic arginine decarboxylases

Biosynthetic arginine decarboxylase (ADC; also known as SpeA) plays an important role in the biosynthesis of polyamines from arginine in bacteria and plants. SpeA is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and shares weak sequence homology with several other PLP-dependent decarboxylases. Here, the crystal structure of PLP-bound SpeA from Campylobacter jejuni is reported at 3.0 Å resolution and that of Escherichia coli SpeA in complex with a sulfate ion is reported at 3.1 Å resolution. The structure of the SpeA monomer contains two large domains, an N-terminal TIM-barrel domain followed by a β-sandwich domain, as well as two smaller helical domains. The TIM-barrel andβ-sandwich domains share structural homology with several other PLP-dependent decarboxylases, even though the sequence conservation among these enzymes is less than 25%. A similar tetramer is observed for both C. jejuni and E. coli SpeA, composed of two dimers of tightly associated monomers. The active site of SpeA is located at the interface of this dimer and is formed by residues from the TIM-barrel domain of one monomer and a highly conserved loop in the β-sandwich domain of the other monomer. The PLP cofactor is recognized by hydrogen-bonding, π-stacking and van der Waals interactions.

The Putative Ca2+-Acting Site in ANO1

Ca2+-activated Cl- channels (CaCC) mediate numerous physiological functions including vectorial Cl- movements across epithelia. Anoctamin1/TMEM16A (Ano1) confers Ca2+-activated Cl- currents. ANO1 having TMs with a putative pore region between TM5 and TM6 shows similar biophysical as well as pharmacological properties as those known for endogenous CaCCs. More importantly, ANO1 is activated by intracellular Ca2+ with EC50 of 2.6 microM at −60 mV. Furthermore, ANO1 activation is also voltage dependent as known for endogenous CaCCs. Because functional importance lies on its activation by intracellular Ca2+, the Ca2+ action site in ANO1 was determined with mutagesis. However, unlike other Ca2+-activated channels such as BK channels, there is no consensus sites for Ca2+ binding except one region that shows weak sequence homology with the Ca2+ action site in BK channel. This region contains many negatively charged amino acids. When we deleted 14 amino acids including the highly negatively charged region, the mutant ANO1 was rarely activated by intracellular Ca2+ with right shift of G-V curves, indicating that this region is important for Ca2+ action. With various mutants in this region, we can localize a sensitive site for Ca2+ response. However, when negatively charged amino acids were replaced by alanine, this mutant showed a comparable sensitivity to Ca2+. Judging from the experimental results of chimera studies with other ANOs, we can conclude that Ca2+ action on this site is essential for its activation.This work was supproted by the WCU program of the Ministry of Education and Science ans Technology of Korea.

Signaling at the membrane interface by the DGK/SK enzyme family

The sphingosine (SK) and diacylglycerol (DGK) kinases have become the subject of considerable focus recently due to their involvement as signaling enzymes in a variety of important biological processes. These lipid signaling kinases are closely related by sequence as well as functional properties. These enzymes are soluble, yet their substrates are hydrophobic. Therefore, they must act at the membrane interface. Second, for both of these enzyme families, their substrates (diacylglycerol for DGKs, sphingosine for SKs) as well as their products (phosphatidic acid for DGK, sphingosine-1-phosphate for SK) have signaling function. To understand how the signaling processes emanating from these kinases are regulated it is critical to understand the fundamental mechanisms that control their enzymatic activity. This is particularly true for the rational design of small molecules that would be useful as therapeutic compounds. Here we summarize enzymological properties of the diacylglycerol and SKs. Further, because the three-dimensional structure of the eukaryotic members of this family has yet to be determined, we discuss what can be gleaned from the recently reported structures of related prokaryotic members of this enzyme family.

PP4R4/KIAA1622 Forms a Novel Stable Cytosolic Complex with Phosphoprotein Phosphatase 4

Protein serine/threonine phosphatase 4 (PP4c) is an essential polypeptide involved in critical cellular processes such as microtubule growth and organization, DNA damage checkpoint recovery, apoptosis, and tumor necrosis factor alpha signaling. Like other phosphatases of the PP2A family, PP4c interacts with regulatory proteins, which specify substrate targeting and intracellular localization. The identification of these regulatory proteins is, therefore, key to fully understanding the function of this enzyme class. Here, using a sensitive affinity purification/mass spectrometry approach, we identify a novel, stable cytosolic PP4c interacting partner, KIAA1622, which we have renamed PP4R4. PP4R4 displays weak sequence homology with the A (scaffolding) subunit of the PP2A holoenzyme and specifically associates with PP4c (and not with the related PP2Ac or PP6c phosphatases). The PP4c.PP4R4 interaction is disrupted by mutations analogous to those abrogating the association of PP2Ac with PP2A A subunit. However, unlike the PP2A A subunit, which plays a scaffolding role, PP4R4 does not bridge PP4c with previously characterized PP4 regulatory subunits. PP4c.PP4R4 complexes exhibit phosphatase activity toward a fluorogenic substrate and gammaH2AX, but this activity is lower than that associated with the PP4c.PP4R2.PP4R3 complex, which itself is less active than the free PP4c catalytic subunit. Our data demonstrate that PP4R4 forms a novel cytosolic complex with PP4c, independent from the complexes containing PP4R1, PP4R2.PP4R3, and alpha4, and that the regulatory subunits of PP4c have evolved different modes of interaction with the catalytic subunit.

Structures of the Shigella flexneri Type 3 Secretion System Protein MxiC Reveal Conformational Variability Amongst Homologues

Many Gram-negative pathogenic bacteria use a complex macromolecular machine, known as the type 3 secretion system (T3SS), to transfer virulence proteins into host cells. The T3SS is composed of a cytoplasmic bulb, a basal body spanning the inner and outer bacterial membranes, and an extracellular needle. Secretion is regulated by both cytoplasmic and inner membrane proteins that must respond to specific signals in order to ensure that virulence proteins are not secreted before contact with a eukaryotic cell. This negative regulation is mediated, in part, by a family of proteins that are thought to physically block the entrance to the secretion apparatus until an appropriate signal is received following host cell contact. Despite weak sequence homology between proteins of this family, the crystal structures of Shigella flexneri MxiC we present here confirm the conservation of domain topology with the homologue from Yersinia sp. Interestingly, comparison of the Shigella and Yersinia structures reveals a significant structural change that results in substantial domain re-arrangement and opening of one face of the molecule. The conservation of a negatively charged patch on this face suggests it may have a role in binding other components of the T3SS.

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

RNA Polymerase I: A Multifunctional Molecular Machine

In this issue, Kuhn et al. (2007) report the complete structure of the 14-subunit yeast RNA polymerase (Pol) I enzyme at 12 A resolution using cryo-electron microscopy (cryo-EM). Their study reveals that three subunits of Pol I perform functions in transcription elongation that are outsourced to the transcription factors TFIIF and TFIIS in the analogous Pol II transcription system.

Molecular cloning and functional analysis of a novel oncogene, cancer-upregulated gene 2 ( CUG2)

We examined genome-wide differences in gene expression between tumor biopsies and normal tissues in order to identify differentially regulated genes in tumors. Cancer-upregulated gene 2 ( CUG2) was identified as an expressed sequence tag (EST) that exhibits significant differential expression in multiple human cancer types. CUG2 showed weak sequence homology with the down-regulator of transcription 1 ( DR1) gene, a human transcription repressor. We found that EGFP-CUG2 fusion proteins were predominantly localized in the nucleus, suggesting their putative role in gene regulation. In addition, CUG2-overexpressing mouse fibroblast cells exhibited distinct cancer-specific phenotypes in vitro and developed into tumors in nude mice. Taken together, these findings suggest that CUG2 is a novel tumor-associated gene that is commonly activated in various human cancers and exhibits high transforming activities; it possibly belongs to a transcription regulator family that is involved in tumor biogenesis.

ASH structure alignment package: Sensitivity and selectivity in domain classification

BackgroundStructure alignment methods offer the possibility of measuring distant evolutionary relationships between proteins that are not visible by sequence-based analysis. However, the question of how structural differences and similarities ought to be quantified in this regard remains open. In this study we construct a training set of sequence-unique CATH and SCOP domains, from which we develop a scoring function that can reliably identify domains with the same CATH topology and SCOP fold classification. The score is implemented in the ASH structure alignment package, for which the source code and a web service are freely available from the PDBj website .ResultsThe new ASH score shows increased selectivity and sensitivity compared with values reported for several popular programs using the same test set of 4,298,905 structure pairs, yielding an area of .96 under the receiver operating characteristic (ROC) curve. In addition, weak sequence homologies between similar domains are revealed that could not be detected by BLAST sequence alignment. Also, a subset of domain pairs is identified that exhibit high similarity, even though their CATH and SCOP classification differs. Finally, we show that the ranking of alignment programs based solely on geometric measures depends on the choice of the quality measure.ConclusionASH shows high selectivity and sensitivity with regard to domain classification, an important step in defining distantly related protein sequence families. Moreover, the CPU cost per alignment is competitive with the fastest programs, making ASH a practical option for large-scale structure classification studies.

Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori α-(1,3/1,4)-Fucosyltransferases

The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.

XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.