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1.
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
2.
Modes of interaction among yeast Nej1, Lif1 and Dnl4 proteins and comparison to human XLF, XRCC4 and Lig4 总被引:2,自引:0,他引:2
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
3.
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
4.
Yano K Morotomi-Yano K Akiyama H 《The international journal of biochemistry & cell biology》2009,41(6):1237-1240
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
5.
Ma Y Lu H Tippin B Goodman MF Shimazaki N Koiwai O Hsieh CL Schwarz K Lieber MR 《Molecular cell》2004,16(5):701-713
Nonhomologous end joining (NHEJ) is a major pathway in multicellular eukaryotes for repairing double-strand DNA breaks (DSBs). Here, the NHEJ reactions have been reconstituted in vitro by using purified Ku, DNA-PK(cs), Artemis, and XRCC4:DNA ligase IV proteins to join incompatible ends to yield diverse junctions. Purified DNA polymerase (pol) X family members (pol mu, pol lambda, and TdT, but not pol beta) contribute to junctional additions in ways that are consistent with corresponding data from genetic knockout mice. The pol lambda and pol mu contributions require their BRCT domains and are both physically and functionally dependent on Ku. This indicates a specific biochemical function for Ku in NHEJ at incompatible DNA ends. The XRCC4:DNA ligase IV complex is able to ligate one strand that has only minimal base pairing with the antiparallel strand. This important aspect of the ligation leads to an iterative strand-processing model for the steps of NHEJ. 相似文献
6.
Enriqueta Riballo Lisa Woodbine Thomas Stiff Sarah A. Walker Aaron A. Goodarzi Penny A. Jeggo 《Nucleic acids research》2009,37(2):482-492
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
7.
Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism 总被引:2,自引:1,他引:1
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Herrmann G Kais S Hoffbauer J Shah-Hosseini K Brüggenolte N Schober H Fäsi M Schär P 《Nucleic acids research》2007,35(7):2321-2332
The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR. 相似文献
8.
A predominant pathway implicated in repair of DNA double-strand breaks (DSBs) is the evolutionarily conserved non-homologous end-joining (NHEJ) pathway. Among the major constituents of this pathway in Saccharomyces cerevisiae is Nej1p, for which a biochemical function has yet to be determined. In this work we demonstrate that Nej1p exhibits a DNA binding activity (KD approximately 1.8 microM) comparable to Lif1p. Although binding is enhanced with larger substrates (>300 bp), short approximately 20 bp substrates can suffice. This DNA binding activity is the first biochemical evidence supporting the idea that Nej1p plays a direct role in the repair of double-strand breaks. The C-terminus of Nej1p is required for interaction with Lif1p and is sufficient for DNA binding. Structural characterization reveals that Nej1p exists as a dimer, and that residues 1-244 are sufficient for dimer formation. Nej1p (aa 1-244) is shown to be defective in end-joining in vivo. Preliminary functional and structural studies on the Nej1p-Lif1p complex suggest that the proteins stably co-purify and the complex binds DNA with a higher affinity than each independent component. The significance of these results is discussed with reference to current literature on Nej1p and other end-joining factors (mammalian and yeast), specifically the recently identified putative mammalian homologue of Nej1p, XLF/Cernunnos. 相似文献
9.
Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase 总被引:1,自引:0,他引:1
The DNA-dependent protein kinase (DNA-PK), consisting of Ku and the DNA-PK catalytic subunit (DNA-PKcs), and the DNA ligase IV-XRCC4 complex function together in the repair of DNA double-strand breaks by non-homologous end joining. These protein complexes are also required for the completion of V(D)J recombination events in immune cells. Here we demonstrate that the DNA ligase IV-XRCC4 complex binds specifically to the ends of duplex DNA molecules and can act as a bridging factor, linking together duplex DNA molecules with complementary but non-ligatable ends. Although the DNA end-binding protein Ku inhibited DNA joining by DNA ligase IV-XRCC4, it did not prevent this complex from binding to DNA. Instead, DNA ligase IV-XRCC4 and Ku bound simultaneously to the ends of duplex DNA molecules. DNA ligase IV-XRCC4 and DNA-PKcs also formed complexes at the ends of DNA molecules, but DNA-PKcs did not inhibit ligation. Interestingly, DNA-PKcs stimulated intermolecular ligation by DNA ligase IV-XRCC4. In the presence of DNA-PK, the majority of the joining events catalyzed by DNA ligase IV-XRCC4 were intermolecular because Ku inhibited intramolecular ligation, but DNA-PKcs still stimulated intramolecular ligation. We suggest that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation. 相似文献
10.
《DNA Repair》2014
Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: (1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), (2) lesion processing, and (3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. 相似文献
11.
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
12.
DNA double-strand breaks (DSBs) induced in the genome of higher eukaryotes by ionizing radiation (IR) are predominantly removed by two pathways of non-homologous end-joining (NHEJ) termed D-NHEJ and B-NHEJ. While D-NHEJ depends on the activities of the DNA-dependent protein kinase (DNA-PK) and DNA ligase IV/XRCC4/XLF, B-NHEJ utilizes, at least partly, DNA ligase III/XRCC1 and PARP-1. Using in vitro end-joining assays and protein fractionation protocols similar to those previously applied for the characterization of DNA ligase III as an end-joining factor, we identify here histone H1 as an additional putative NHEJ factor. H1 strongly enhances DNA-end joining and shifts the product spectrum from circles to multimers. While H1 enhances the DNA-end-joining activities of both DNA Ligase IV and DNA Ligase III, the effect on ligase III is significantly stronger. Histone H1 also enhances the activity of PARP-1. Since histone H1 has been shown to counteract D-NHEJ, these observations and the known functions of the protein identify it as a putative alignment factor operating preferentially within B-NHEJ. 相似文献
13.
《Cell cycle (Georgetown, Tex.)》2013,12(10):1321-1325
XLF, also known as Cernunnos, is a newly identified core factor of the non-homologous end-joining (NHEJ) pathway for DNA double-strand breaks (DSBs) repair. XLF is known to stimulate DNA ligase IV in vitro through its interaction with XRCC4. Here, we outline the key findings on the dynamic behavior of XLF and XRCC4 at DSBs in living cells. XLF is quickly recruited to DSBs in the absence of XRCC4 or DNA-PKcs. The recruited XLF molecules constantly exchange at DSBs, and XRCC4 modulates the exchange rate of the recruited XLF. XRCC4 can be recruited to DSBs without DNA-PKcs, but DNA-PKcs stabilizes the recruited XRCC4. These observations are inconsistent with the prevailing concept that NHEJ proteins are sequentially recruited to DSBs, which is mainly supported by in vitro evidence. We propose a novel two-phase model for the assembly of NHEJ factors to DSBs in vivo. XLF, XRCC4, and DNA-PKcs are independently recruited to Ku-bound DSBs. The recruited factors are assembled into a large complex, in which the protein interactions observed in vitro define the stability of the recruited factors. This new view has broad implications for the mechanism of DSB sensing and functional protein assembly in the NHEJ pathway. 相似文献
14.
DNA double-strand breaks (DSBs), which are generated by ionizing radiation (IR) and a range of other DNA damaging agents, are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Previous studies have shown that NHEJ in Saccharomyces cerevisiae requires the Yku70p-Yku80p heterodimer and a complex consisting of DNA Ligase IV, Lif1p and Nej1p. Here, we report that Nej1p is phosphorylated in response to DNA damage in a manner that relies on the DNA damage checkpoint kinases Mec1p, Rad53p and Dun1p. By using a mutational approach, we have identified a consensus Dun1p phosphorylation site in Nej1p, and mutation of conserved serine residues within it leads to decreased NHEJ efficiency. These data, together with previous findings that Rad55p--a protein involved in HR--is phosphorylated analogously, point to there being a broad signalling network connecting DNA damage checkpoint responses with the regulation of DNA DSB repair activities. 相似文献
15.
《DNA Repair》2015
The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3′-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. 相似文献
16.
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
17.
Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here, we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. In contrast, binding of ligase IV to DNA-PKcs or XRCC4 to Ku is very weak or non-existent. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks. 相似文献
18.
Rulten SL Fisher AE Robert I Zuma MC Rouleau M Ju L Poirier G Reina-San-Martin B Caldecott KW 《Molecular cell》2011,41(1):33-45
PARP-3 is a member of the ADP-ribosyl transferase superfamily of unknown function. We show that PARP-3 is stimulated by DNA double-strand breaks (DSBs) in vitro and functions in the same pathway as the poly (ADP-ribose)-binding protein APLF to accelerate chromosomal DNA DSB repair. We implicate PARP-3 in the accumulation of APLF at DSBs and demonstrate that APLF promotes the retention of XRCC4/DNA ligase IV complex in chromatin, suggesting that PARP-3 and APLF accelerate DNA ligation during nonhomologous end-joining (NHEJ). Consistent with this, we show that class switch recombination in Aplf(-/-) B cells is biased toward microhomology-mediated end-joining, a pathway that operates in the absence of XRCC4/DNA ligase IV, and that the requirement for PARP-3 and APLF for NHEJ is circumvented by overexpression of XRCC4/DNA ligase IV. These data identify molecular roles for PARP-3 and APLF in chromosomal DNA double-strand break repair reactions. 相似文献
19.
Howard H. Y. Chang Go Watanabe Christina A. Gerodimos Takashi Ochi Tom L. Blundell Stephen P. Jackson Michael R. Lieber 《The Journal of biological chemistry》2016,291(47):24377-24389
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
20.
In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway. 相似文献