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Nonhomologous end-joining (NHEJ) is an important pathway for the repair of DNA double-strand breaks (DSBs) and plays a critical role in maintaining genomic stability in mammalian cells. While Ku70/80 (Ku) functions in NHEJ as part of the DNA-dependent protein kinase (DNA-PK), genetic evidence indicates that the role of Ku in NHEJ goes beyond its participation in DNA-PK. Inositol hexakisphosphate (IP6) was previously found to stimulate NHEJ in vitro and Ku was identified as an IP6-binding factor. Through mutational analysis, we identified a bipartite IP6-binding site in Ku and generated IP6-binding mutants that ranged from 1.22% to 58.48% of wild-type binding. Significantly, these Ku IP6-binding mutants were impaired for participation in NHEJ in vitro and we observed a positive correlation between IP6 binding and NHEJ. Ku IP6-binding mutants were separation-of-function mutants that bound DNA and activated DNA-PK as well as wild-type Ku. Our observations identify a hitherto undefined IP6-binding site in Ku and show that this interaction is important for DSB repair by NHEJ in vitro. Moreover, these data indicate that in addition to binding of exposed DNA termini and activation of DNA-PK, the Ku heterodimer plays a role in mammalian NHEJ that is regulated by binding of IP6.  相似文献   

3.
We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.  相似文献   

4.

Background  

DNA double-strand breaks (DSBs) can occur in response to ionizing radiation (IR), radiomimetic agents and from endogenous DNA-damaging reactive oxygen metabolites. Unrepaired or improperly repaired DSBs are potentially the most lethal form of DNA damage and can result in chromosomal translocations and contribute to the development of cancer. The principal mechanism for the repair of DSBs in humans is non-homologous end-joining (NHEJ). Ku is a key member of the NHEJ pathway and plays an important role in the recognition step when it binds to free DNA termini. Ku then stimulates the assembly and activation of other NHEJ components. DNA binding of Ku is regulated by redox conditions and evidence from our laboratory has demonstrated that Ku undergoes structural changes when oxidized that results in a reduction in DNA binding activity. The C-terminal domain and cysteine 493 of Ku80 were investigated for their contribution to redox regulation of Ku.  相似文献   

5.
Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 heterodimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3′-overhangs that abut a 5′-overhang or blunt end. While the sequences of the 3′-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.  相似文献   

6.
Rad9是一种重要的细胞周期监控点调控蛋白.越来越多的证据显示,Rad9也可与多种DNA损伤修复通路中的蛋白质相互作用,并调节其功能,在DNA损伤修复中发挥重要作用.非同源末端连接修复是DNA双链断裂的一条重要修复途径.Ku70、Ku80和DNA依赖的蛋白激酶催化亚基(DNA-PKcs)共同组成DNA依赖的蛋白激酶复合物(DNA-PK),在非同源末端修复连接中起重要作用.本研究中,检测到Rad9与Ku70有直接的物理相互作用和功能相互作用.我们在不同的细胞模型中发现,Rad9基因敲除、Rad9蛋白去除或Rad9表达降低会导致非同源末端连接效率明显下降.已有的研究表明,DNA损伤可导致细胞中Ku70与染色质结合增加及DNA-PKcs激酶活性增强.我们的结果显示,与野生小鼠细胞相比,Rad9基因敲除的小鼠细胞中, DNA损伤诱导的上述效应均减弱.综上所述,我们的研究首次报道了Rad9与非同源末端连接修复蛋白Ku70间有相互作用,并提示Rad9可通过调节Ku70/Ku80/DNA-PKcs复合物功能参与非同源末端连接修复.  相似文献   

7.
A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA–PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA–PKcs quickly mobilized in response to IR and bleomycin treatments consistent with prior reports using laser-generated DSBs. The response was linearly dependent on IR dose, and blocking NHEJ enhanced immobilization of both Ku80 and DNA–PKcs after DNA damage. These findings support the idea of using Number and Brightness and Raster-scan Image Correlation Spectroscopy as methods to monitor kinetics of DSB repair proteins in living cells under conditions mimicking radiation and chemotherapy treatments.  相似文献   

8.
We have determined the effect of cisplatin–DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro. The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin–DNA adducts compared to control, undamaged duplex DNA. The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku–DNA complex. In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin–DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA. The position of the cisplatin–DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus. These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation. The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus. The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.  相似文献   

9.
Bacterial non-homologous end joining requires the ligase, LigD and Ku. Ku finds the break site, recruits LigD, and then assists LigD to seal the phosphodiester backbone. Bacterial Ku contains a core domain conserved with eukaryotes but has a unique C-terminus that can be divided into a minimal C-terminal region that is conserved and an extended C-terminal region that varies in sequence and length between species. Here, we examine the role of Mycobacterium tuberculosis Ku C-terminal variants, where we removed either the extended or entire C-terminus to investigate the effects on Ku–DNA binding, rates of Ku-stimulated ligation, and binding affinity of a direct Ku–LigD interaction. We find that the extended C-terminus limits DNA binding and identify key amino acids that contribute to this effect through alanine-scanning mutagenesis. The minimal C-terminus is sufficient to stimulate ligation of double-stranded DNA, but the Ku core domain also contributes to stimulating ligation. We further show that wildtype Ku and the Ku core domain alone directly bind both ligase and polymerase domains of LigD. Our results suggest that Ku-stimulated ligation involves direct interactions between the Ku core domain and the LigD ligase domain, in addition to the extended Ku C-terminus and the LigD polymerase domain.  相似文献   

10.
Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4−/− compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs.  相似文献   

11.
The Ku heterodimer, comprised of Ku70 and Ku80 subunits, is a conserved complex involved in nonhomologous end-joining (NHEJ). However, it also functions in maintenance of telomeres, chromosome termini normally resistant to end-joining events. To elucidate the spatial organization of these functions, we rationally guided Ku mutagenesis in yeast with real-valued evolutionary trace (rvET). This revealed two ancestrally related alpha-helices: one on the Ku70 surface that is required in yeast for NHEJ, and a second on the Ku80 surface that is required in yeast for telomeric heterochromatin formation. When bound to a DNA end, the surface containing the NHEJ-specific Ku70 helix is oriented toward the DNA terminus, whereas the surface containing the telomeric function-specific Ku80 helix faces inward, toward telomeric chromatin, when bound to a telomere. We propose a 'two-face' model for Ku and that divergent evolution of these faces allowed Ku's dual role in NHEJ and telomere maintenance.  相似文献   

12.
Hanakahi LA  West SC 《The EMBO journal》2002,21(8):2038-2044
In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.  相似文献   

13.
Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80−/− mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs−/− mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80−/−, dna-pkcs−/− and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PKCS causes reduced lifespan and bodyweights, which is most severe in ku80−/− mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80−/− mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PKCS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PKCS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PKCS are essential for NHEJ.  相似文献   

14.
Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G1 phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G1. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G1 phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G2/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G1.  相似文献   

15.
The Ku heterodimer, a DNA repair protein complex consisting of 70- and 80-kDa subunits, is involved in the non-homologous end-joining (NHEJ) pathway. Plants are thought to use the NHEJ pathway primarily for the repair of DNA double-strand breaks (DSBs). The Ku70/80 protein has been identified in many plants and been shown to possess several similar functions to its counter protein complex in mammals. In the present study, ovate family protein 1 (AtOFP1) was demonstrated to be a plant Ku-interacting protein by yeast two-hybrid screening and the GST pull-down assay. Truncation analysis revealed that the C-terminal domain of AtKu70 contains interacting sites for AtOFP1. The electrophoretic mobility shift assay (EMSA) indicated that AtOFP1 is also a DNA binding protein with its binding domain at the N-terminus. In 3-week-old seedlings, expression of the AtOFP1 gene increased after exposure to DNA-damaging agents (such as methyl methanesulfonate (MMS) and menadione) in a time dependent manner. Seedlings lacking the AtOFP1 protein were more sensitive to MMS and menadione as compared with wild-type. Furthermore, similar to AtKu70 ?/? and AtKu80 ?/?, the AtOFP1 ?/? mutant showed relatively lower NHEJ activity in vivo. Taken together, these results suggest that AtOFP1 may play a role in DNA repair through the NHEJ pathway accompanying with the AtKu protein.  相似文献   

16.
Evolutionary selection ensures specificity and efficiency in dynamic metastable macromolecular machines that repair DNA damage without releasing toxic and mutagenic intermediates. Here we examine non‐homologous end joining (NHEJ) as the primary conserved DNA double‐strand break (DSB) repair process in human cells. NHEJ has exemplary key roles in networks determining the development, outcome of cancer treatments by DSB‐inducing agents, generation of antibody and T‐cell receptor diversity, and innate immune response for RNA viruses. We determine mechanistic insights into NHEJ structural biochemistry focusing upon advanced small angle X‐ray scattering (SAXS) results combined with X‐ray crystallography (MX) and cryo‐electron microscopy (cryo‐EM). SAXS coupled to atomic structures enables integrated structural biology for objective quantitative assessment of conformational ensembles and assemblies in solution, intra‐molecular distances, structural similarity, functional disorder, conformational switching, and flexibility. Importantly, NHEJ complexes in solution undergo larger allosteric transitions than seen in their cryo‐EM or MX structures. In the long‐range synaptic complex, X‐ray repair cross‐complementing 4 (XRCC4) plus XRCC4‐like‐factor (XLF) form a flexible bridge and linchpin for DNA ends bound to KU heterodimer (Ku70/80) and DNA‐PKcs (DNA‐dependent protein kinase catalytic subunit). Upon binding two DNA ends, auto‐phosphorylation opens DNA‐PKcs dimer licensing NHEJ via concerted conformational transformations of XLF‐XRCC4, XLF–Ku80, and LigIVBRCT–Ku70 interfaces. Integrated structures reveal multifunctional roles for disordered linkers and modular dynamic interfaces promoting DSB end processing and alignment into the short‐range complex for ligation by LigIV. Integrated findings define dynamic assemblies fundamental to designing separation‐of‐function mutants and allosteric inhibitors targeting conformational transitions in multifunctional complexes.  相似文献   

17.
Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.  相似文献   

18.
The autoantigen Ku, composed of subunits Ku70 and Ku86, is necessary for repair of DNA double-strand breaks by nonhomologous end joining. Similarly, Ku participates in repair of DNA double-strand breaks that occur during V(D)J recombination, and it is therefore required for the development of B and T lymphocytes. Although previous studies have identified the DNA-binding activities of Ku, little is known concerning its dynamics, such as the mobility of Ku in the nucleus and its rate of association with substrates. To address this question, fluorescence photobleaching experiments were performed using HeLa cells and B cells expressing a green fluorescent protein (GFP) fusion construct of either Ku70 or Ku86. The results show that Ku moves rapidly throughout the nucleus even following irradiation of the cells. However, the rate of diffusion of Ku was approximately 100-fold slower than that predicted from its size. Association of Ku-GFP with a filamentous nuclear structure was also evident, and nuclear extraction experiments suggest that this represents nuclear matrix. A central domain of Ku70 containing its DNA-binding and heterodimerization regions and its nuclear localization signal shows that this alone is sufficient for the observed mobility of Ku70-GFP and its association with nuclear matrix. These data suggest the mobility of Ku is characterized by a transient, high flux association with nuclear substrates that includes both DNA and the nuclear matrix and may represent a mechanism for repair of double-strand breaks using the nuclear matrix as a scaffold.  相似文献   

19.
The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic Escherichia coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (approximately 9000 A2 [J.R. Walker, R.A. Corpina, J. Goldberg, Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair, Nature 412 (2001) 607-614]), which suggests that herterodimerization may be important for protein stability. N-terminally His6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of His6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli.  相似文献   

20.
MSH6, a key component of the MSH2-MSH6 complex, plays a fundamental role in the repair of mismatched DNA bases. Herein, we report that MSH6 is a novel Ku70-interacting protein identified by yeast two-hybrid screening. Ku70 and Ku86 are two key regulatory subunits of the DNA-dependent protein kinase, which plays an essential role in repair of DNA double-strand breaks (DSBs) through the non-homologous end-joining (NEHJ) pathway. We found that association of Ku70 with MSH6 is enhanced in response to treatment with the radiomimetic drug neocarzinostatin (NCS) or ionizing radiation (IR), a potent inducer of DSBs. Furthermore, MSH6 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci after NCS or IR treatment. MSH6 colocalizes with γ-H2AX at sites of DNA damage after NCS or IR treatment. Cells depleted of MSH6 accumulate high levels of persistent DSBs, as detected by formation of γ-H2AX foci and by the comet assay. Moreover, MSH6-deficient cells were also shown to exhibit impaired NHEJ, which could be rescued by MSH6 overexpression. MSH6-deficient cells were hypersensitive to NCS- or IR-induced cell death, as revealed by a clonogenic cell-survival assay. These results suggest a potential role for MSH6 in DSB repair through upregulation of NHEJ by association with Ku70.  相似文献   

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