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1.
The XRCC4-like factor (XLF)-XRCC4 complex is essential for nonhomologous end joining, the major repair pathway for DNA double strand breaks in human cells. Yet, how XLF binds XRCC4 and impacts nonhomologous end joining functions has been enigmatic. Here, we report the XLF-XRCC4 complex crystal structure in combination with biophysical and mutational analyses to define the XLF-XRCC4 interactions. Crystal and solution structures plus mutations characterize alternating XRCC4 and XLF head domain interfaces forming parallel super-helical filaments. XLF Leu-115 ("Leu-lock") inserts into a hydrophobic pocket formed by XRCC4 Met-59, Met-61, Lys-65, Lys-99, Phe-106, and Leu-108 in synergy with pseudo-symmetric β-zipper hydrogen bonds to drive specificity. XLF C terminus and DNA enhance parallel filament formation. Super-helical XLF-XRCC4 filaments form a positively charged channel to bind DNA and align ends for efficient ligation. Collective results reveal how human XLF and XRCC4 interact to bind DNA, suggest consequences of patient mutations, and support a unified molecular mechanism for XLF-XRCC4 stimulation of DNA ligation.  相似文献   

2.
The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.  相似文献   

3.
In mammalian cells, non-homologous end joining (NHEJ) is the major double strand break (DSB) repair mechanism during the G(1) phase of the cell cycle. It also contributes to DSB repair during the S and G(2) phases. Ku heterodimer, DNA PKcs, XRCC4 and DNA Ligase IV constitute the core NHEJ machinery, which joins directly ligatable ends. XRCC4-like factor/Cernunnos (XLF/Cer) is a recently discovered interaction partner of XRCC4. Current evidence suggests the following model for the role of XLF/Cer in NHEJ: after DSB induction, the XRCC4-DNA Ligase IV complex promotes efficient accumulation of XLF/Cer at DNA damage sites via constitutive interaction of the XRCC4 and XLF/Cer head domains and dependent on components of the DNA PK complex. Ku alone can stabilise the association of XLF/Cer with DNA ends. XLF/Cer stimulates ligation of complementary and non-complementary DNA ends by XRCC4-DNA Ligase IV. This activity involves the carboxy-terminal DNA binding region of XLF/Cer and could occur via different, non-exclusive modes: (i) enhancement of the stability of the XRCC4-DNA Ligase IV complex on DNA ends by XLF/Cer, (ii) modulation of the efficiency and/or specificity of DNA Ligase IV by binding of XLF/Cer to the XRCC4-DNA Ligase IV complex, (iii) promotion of the alignment of blunt or other non-complementary DNA ends by XLF/Cer for ligation. XLF/Cer promotes the preservation of 3' overhangs, restricts nucleotide loss and thereby promotes accuracy of DSB joining by XRCC4-DNA Ligase IV during NHEJ and V(D)J recombination.  相似文献   

4.
Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.Double-stranded DNA breaks (DSBs) are among the most cytotoxic DNA lesions for mammalian cells.1 Effective repair of DSBs is essential for cellular survival and for suppression of potential deleterious chromosomal rearrangements.2 Two main DNA repair pathways eliminate DSBs—homologous recombination (HR) or non-homologous end joining (NHEJ). HR utilises an undamaged copy of the chromosome as a template to direct repair, thus this restricts HR to the S and G2/M phases of the cell cycle, when such an extra chromosome copy is available.3 NHEJ performs the bulk of DSB repair in mammalian cells and in particular in during the G1 phase of the cell cycle, where the cells are completely dependent on NHEJ. NHEJ can be further subdivided into so-called classical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ).4 These DNA repair pathways utilise distinct protein components and also show different efficiencies of end ligation. In general, c-NHEJ is much more effective in end ligation than alt-NHEJ and can ligate most unrelated DNA ends directly or with minimal processing. In contrast alt-NHEJ requires short microhomologies between the DNA ends for ligation.5 C-NHEJ requires the following seven core proteins: Ku70/Ku86 dimers, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis nuclease, XRCC4-like factor (XLF) and the XRCC4/ligase IV complex.6, 7 The DSB repair during c-NHEJ is initiated by the Ku dimer that senses the presence of free double-stranded DNA ends in cells and rapidly binds such ends with high affinity. DNA-bound Ku then recruits DNA-PKcs (DNA-PKcs/Ku70/Ku86 complex is termed DNA-PK holoenzyme), which has a protein kinase activity and is required for activation of the nuclease Artemis.8 Artemis, in turn, is responsible for DNA end processing in order to achieve DNA end structures suitable for ligation. The final step of c-NHEJ is the ligation of processed DNA ends by XRCC4/ligase IV complex. This final step is stimulated by XLF protein that interacts with XRCC4 forming long filamentous structures at DSBs to facilitate DNA end joining.9, 10 XRCC4 and XLF factors are distinct among NHEJ factors in that they share similar tertiary structure but show low primary sequence conservation.11 Since the identification of XLF in 2006, no new core factors have been discovered.11, 12 Importantly, c-NHEJ is essential for proper development, as mutations in this pathway lead to immunodeficiency and defective neurogenesis in humans.7 It is therefore essential to fully decipher the identity of components for the c-NHEJ pathway and their regulation.In this study, proteomic analysis of DNA-PKcs-containing protein complexes identified an abundant previously uncharacterised protein c9orf142, which we have named c9orf142—XLS (XRCC4-like small protein). Structural modelling predicts XLS to be highly similar to XRCC4 and XLF, and depletion of XLS delays ionising radiation (IR)-induced DNA DSB repair. Moreover, XLS is associated with other core c-NHEJ factors. Our data strongly suggest that c9orf142/XLS represents a novel c-NHEJ component in mammalian cells.  相似文献   

5.
The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.  相似文献   

6.
DNA ligase IV (LigIV) is critical for nonhomologous 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 small angle 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.  相似文献   

7.
XLF-Cernunnos (XLF) is a component of the DNA ligase IV–XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.  相似文献   

8.
XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together--stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.  相似文献   

9.
Crystal structure of human XLF: a twist in nonhomologous DNA end-joining   总被引:3,自引:0,他引:3  
Andres SN  Modesti M  Tsai CJ  Chu G  Junop MS 《Molecular cell》2007,28(6):1093-1101
DNA double-strand breaks represent one of the most severe forms of DNA damage in mammalian cells. One pathway for repairing these breaks occurs via nonhomologous end-joining (NHEJ) and depends on XRCC4, LigaseIV, and Cernunnos, also called XLF. Although XLF stimulates XRCC4/LigaseIV to ligate mismatched and noncohesive DNA ends, the mechanistic basis for this function remains unclear. Here we report the structure of a partially functional 224 residue N-terminal fragment of human XLF. Despite only weak sequence similarity, XLF(1-170) shares structural homology with XRCC4(1-159). However, unlike the highly extended 130 A helical domain observed in XRCC4, XLF adopts a more compact, folded helical C-terminal region involving two turns and a twist, wrapping back to the structurally conserved N terminus. Mutational analysis of XLF and XRCC4 reveals a potential interaction interface, suggesting a mechanism for how XLF stimulates the ligation of mismatched ends.  相似文献   

10.
An XRCC4-like factor, called XLF or Cernunnos, was recently identified as another important factor in the non-homologous DNA end joining (NHEJ) process. NHEJ is the major pathway for the repair of double-strand DNA breaks. The similarity in the putative secondary structures of XLF and XRCC4 as well as the association of XLF with XRCC4.DNA ligase IV in vivo suggested a role in the final ligation step of NHEJ. Here, we find that purified XLF directly interacts with purified XRCC4.DNA ligase IV complex and stimulates the ligase complex in a direct assay for ligation activity. Purified XLF has DNA binding activity, but this binding is dependent on DNA length in a manner most consistent with orientation of the C-terminal alpha helices parallel to the DNA helix. To better understand the function of XLF, we purified an XLF mutant (R57G), which was identified in patients with NHEJ deficiency and severe combined immunodeficiency. Surprisingly, the mutant protein retained its ability to stimulate XRCC4.DNA ligase IV but failed to translocate to the nucleus, and this appears to be the basis for the NHEJ defect in this patient.  相似文献   

11.
Osteosarcoma (OS) is the most common primary malignant bone tumor diagnosed in children and adolescents. Unfortunately, OS patients with metastatic or recurrent disease are highly resistant to front line chemotherapy that significantly limits the long-term survival rate. Since majority of chemotherapeutic agents used in OS work by generating DNA damages, enhanced DNA repair pathways are generally associated with chemoresistance in OS treatment. However, the exact mechanisms of chemoresistance in OS are not fully understood. Our study found that paralogue of XRCC4 and XLF (PAXX), which was identified recently as a novel factor of non-homologous end joining (NHEJ), is upregulated in chemoresistant OS cells. Importantly, PAXX deficiency re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Mechanistically, chemoresistance to doxorubicin or cisplatin results in enhanced PAXX-Ku70 interaction and elevated NHEJ efficiency. We also identified a small molecule M11 that interrupts PAXX-Ku70 interaction and re-sensitizes chemoresistant OS cells to doxorubicin and cisplatin. Thus, our data provide evidence that PAXX could serve as a novel target to overcome chemoresistance in OS treatment.  相似文献   

12.
Aprataxin polynucleotide kinase/phosphatase-like factor (APLF) facilitates nonhomologous end joining (NHEJ) and associates with the core NHEJ components XRCC4-DNA ligase IV and Ku. The APLF forkhead-associated (FHA) domain directs interactions with XRCC4, but the APLF-Ku interaction has not been well characterized. Here we describe an evolutionarily conserved amino acid motif within APLF that is required for mediating the physical interaction between APLF and Ku. This APLF Ku-binding motif possesses a similarity to regions identified in other NHEJ factors, WRN and XLF, which also direct interactions with Ku. Indeed, peptides derived from the Ku-binding region of APLF, WRN, or XLF were sufficient to reconstitute the interaction with Ku in vitro. Although APLF is localized predominantly to the nucleus, it does not possess a nuclear localization signal (NLS). Interestingly, the disruption of the APLF-Ku interaction by substituting key residues in the APLF Ku-binding motif was associated with increased relocalization of APLF to the cytoplasm and reduced association with XRCC4, which was rescued by the introduction of an NLS onto APLF. When human cells stably depleted of APLF were reconstituted with APLF Ku-binding mutants, or with an APLF FHA mutant that is known to disrupt interactions with XRCC4, APLF-dependent NHEJ and the retention of APLF at sites of laser-generated DNA damage were impaired. These data suggest functional requirements for Ku and XRCC4 in APLF-dependent NHEJ and a unique role for Ku as a factor required to facilitate the nuclear retention of APLF.  相似文献   

13.
DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4–XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4–XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4–XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4–XLF at 3.94 Å, a model for XRCC4–XLF complex function in NHEJ is presented.  相似文献   

14.
Yu Y  Mahaney BL  Yano K  Ye R  Fang S  Douglas P  Chen DJ  Lees-Miller SP 《DNA Repair》2008,7(10):1680-1692
Nonhomologous end joining (NHEJ) is the major pathway for the repair of DNA double strand breaks (DSBs) in human cells. NHEJ requires the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku70, Ku80, XRCC4, DNA ligase IV and Artemis, as well as DNA polymerases mu and lambda and polynucleotide kinase. Recent studies have identified an additional participant, XLF, for XRCC4-like factor (also called Cernunnos), which interacts with the XRCC4-DNA ligase IV complex and stimulates its activity in vitro, however, its precise role in the DNA damage response is not fully understood. Since the protein kinase activity of DNA-PKcs is required for NHEJ, we asked whether XLF might be a physiological target of DNA-PK. Here, we have identified two major in vitro DNA-PK phosphorylation sites in the C-terminal region of XLF, serines 245 and 251. We show that these represent the major phosphorylation sites in XLF in vivo and that serine 245 is phosphorylated in vivo by DNA-PK, while serine 251 is phosphorylated by Ataxia-Telangiectasia Mutated (ATM). However, phosphorylation of XLF did not have a significant effect on the ability of XLF to interact with DNA in vitro or its recruitment to laser-induced DSBs in vivo. Similarly, XLF in which the identified in vivo phosphorylation sites were mutated to alanine was able to complement the DSB repair defect as well as radiation sensitivity in XLF-deficient 2BN cells. We conclude that phosphorylation of XLF at these sites does not play a major role in the repair of IR-induced DSBs in vivo.  相似文献   

15.
Inositol hexakisphosphate (InsP(6)) is a member of the inositol polyphosphate group that participates in numerous intracellular signaling pathways. Cheung and colleagues previously reported that InsP(6) stimulated double-strand break repair by nonhomologous end joining (NHEJ) in cell-free extracts and that InsP(6) binding by the Ku70/80 subunit of the DNA-dependent protein kinase (DNA-PK) was required for stimulation of NHEJ in vitro. This report describes InsP(6)-dependent phosphorylation of two NHEJ factors, XRCC4 and XLF, in partially purified human cell extracts. XRCC4 and XLF are known substrates for DNA-PK, which does not require InsP(6) for protein kinase activity. Consistent with a role for DNA-PK in these reactions, InsP(6)-dependent phosphorylation of XRCC4 and XLF was DNA dependent and not observed in the presence of DNA-PK inhibitors. Depletion of the Ku70/80 DNA-, InsP(6)-binding subunit of DNA-PK resulted in loss of InsP(6)-dependent phosphorylation and showed a requirement for Ku70/80 in these reactions. Complementation of Ku70/80-depleted reactions with recombinant wild-type Ku70/80 restored InsP(6)-dependent phosphorylation of XRCC4 and XLF. In contrast, addition of a Ku70/80 mutant with reduced InsP(6) binding failed to restore InsP(6)-dependent phosphorylation. While additional protein kinases may participate in InsP(6)-dependent phosphorylation of XRCC4 and XLF, data presented here describe a clear requirement for DNA-PK in these phosphorylation events. Furthermore, these data suggest that binding of the inositol polyphosphate InsP(6) by Ku70/80 may modulate the substrate specificity of the phosphoinositide-3-kinase-related protein kinase DNA-PK.  相似文献   

16.
Non‐homologous end joining (NHEJ) is critical for the maintenance of genetic integrity and DNA double‐strand break (DSB) repair. NHEJ is regulated by a series of interactions between core components of the pathway, including Ku heterodimer, XLF/Cernunnos, and XRCC4/DNA Ligase 4 (Lig4). However, the mechanisms by which these proteins assemble into functional protein–DNA complexes are not fully understood. Here, we show that the von Willebrand (vWA) domain of Ku80 fulfills a critical role in this process by recruiting Aprataxin‐and‐PNK‐Like Factor (APLF) into Ku‐DNA complexes. APLF, in turn, functions as a scaffold protein and promotes the recruitment and/or retention of XRCC4‐Lig4 and XLF, thereby assembling multi‐protein Ku complexes capable of efficient DNA ligation in vitro and in cells. Disruption of the interactions between APLF and either Ku80 or XRCC4‐Lig4 disrupts the assembly and activity of Ku complexes, and confers cellular hypersensitivity and reduced rates of chromosomal DSB repair in avian and human cells, respectively. Collectively, these data identify a role for the vWA domain of Ku80 and a molecular mechanism by which DNA ligase proficient complexes are assembled during NHEJ in mammalian cells, and reveal APLF to be a structural component of this critical DSB repair pathway.  相似文献   

17.
Non-homologous end-joining (NHEJ) is the predominant repair pathway for DNA double-strand breaks (DSBs) in vertebrates and also plays a crucial role in V(D)J recombination of immunoglobulin genes. Cernunnos/XLF is a newly identified core factor for NHEJ, and its defect causes a genetic disease characterized by neural disorders, immunodeficiency and increased radiosensitivity. Cernunnos/XLF has at least two distinct functions in NHEJ. Cernunnos/XLF interacts with and stimulates the XRCC4/DNA ligase IV complex, which acts at the final ligation step in NHEJ. In living cells, Cernunnos/XLF quickly responds to DSB induction and accumulates at damaged sites in a Ku-dependent but XRCC4-independent manner. These observations indicate that Cernunnos/XLF plays a unique role in bridging damage sensing and DSB rejoining steps of NHEJ. Recent crystallographic analyses of the homodimeric Cernunnos/XLF protein provide structural insights into the Cernunnos/XLF functions. These studies offer important clues toward understanding the molecular mechanism for NHEJ-defective diseases.  相似文献   

18.
Ku recruits XLF to DNA double-strand breaks   总被引:3,自引:0,他引:3  
XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.  相似文献   

19.
Deshpande RA  Wilson TE 《DNA Repair》2007,6(10):1507-1516
The nonhomologous end joining (NHEJ) pathway of double-strand break repair depends on DNA ligase IV and its interacting partner protein XRCC4 (Lif1 in yeast). A third yeast protein, Nej1, interacts with Lif1 and supports NHEJ, similar to the distantly related mammalian Nej1 orthologue XLF (also known as Cernunnos). XRCC4/Lif1 and XLF/Nej1 are themselves related and likely fold into similar coiled-coil structures, which suggests many possible modes of interaction between these proteins. Using yeast two-hybrid and co-precipitation methods we examined these interactions and the protein domains required to support them. Results suggest that stable coiled-coil homodimers are a predominant form of XLF/Nej1, just as for XRCC4/Lif1, but that similar heterodimers are not. XLF-XRCC4 and Nej1-Lif1 interactions were instead mediated independently of the coiled coil, and by different regions of XLF and Nej1. Specifically, the globular head of XRCC4/Lif1 interacted with N- and C-terminal domains of XLF and Nej1, respectively. Direct interactions between XLF/Nej1 and DNA ligase IV were also observed, but again appeared qualitatively different than the stable coiled-coil-mediated interaction between XRCC4/Lif1 and DNA ligase IV. The implications of these findings for DNA ligase IV function are considered in light of the evolutionary pattern in the XLF/XRCC4 and XLF/Nej1 family.  相似文献   

20.
Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3′ overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, whereas XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to five molecules each of XL and XLF into a Ku-XL-XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL, and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to six molecules each of Ku and XL into a Ku-XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular mass, MEnd ligase-DNA had a lower electrophoretic mobility than Ku-XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggests that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation.  相似文献   

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