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1.
Maria Quanz Danielle Chassoux Nathalie Berthault Céline Agrario Jian-Sheng Sun Marie Dutreix 《PloS one》2009,4(7)
Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an “all-or-none” pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a “false” DNA damage signaling that disorganizes the DNA repair system. 相似文献
2.
Amelie Croset Fabrice P. Cordelières Nathalie Berthault Cyril Buhler Jian-Sheng Sun Maria Quanz Marie Dutreix 《Nucleic acids research》2013,41(15):7344-7355
One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations. 相似文献
3.
Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication. 总被引:8,自引:2,他引:6
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The single-stranded DNA-binding protein, Replication Protein A (RPA), is a heterotrimeric complex with subunits of 70, 32 and 14 kDa involved in DNA metabolism. RPA may be a target for cellular regulation; the 32 kDa subunit (RPA32) is phosphorylated by several cellular kinases including the DNA-dependent protein kinase (DNA-PK). We have purified a mutant hRPA complex lacking amino acids 1-33 of RPA32 (rhRPA x 32delta1-33). This mutant bound ssDNA and supported DNA replication; however, rhRPA x 32delta1-33 was not phosphorylated under replication conditions or directly by DNA-PK. Proteolytic mapping revealed that all the sites phosphorylated by DNA-PK are contained on residues 1-33 of RPA32. When wild-type RPA was treated with DNA-PK and the mixture added to SV40 replication assays, DNA replication was supported. In contrast, when rhRPA x 32delta1-33 was treated with DNA-PK, DNA replication was strongly inhibited. Because untreated rhRPA x 32delta1-33 is fully functional, this suggests that the N-terminus of RPA is needed to overcome inhibitory effects of DNA-PK on other components of the DNA replication system. Thus, phosphorylation of RPA may modulate DNA replication indirectly, through interactions with other proteins whose activity is modulated by phosphorylation. 相似文献
4.
O'Neill T Dwyer AJ Ziv Y Chan DW Lees-Miller SP Abraham RH Lai JH Hill D Shiloh Y Cantley LC Rathbun GA 《The Journal of biological chemistry》2000,275(30):22719-22727
The ataxia telangiectasia mutated (ATM) gene encodes a serine/threonine protein kinase that plays a critical role in genomic surveillance and development. Here, we use a peptide library approach to define the in vitro substrate specificity of ATM kinase activity. The peptide library analysis identified an optimal sequence with a central core motif of LSQE that is preferentially phosphorylated by ATM. The contributions of the amino acids surrounding serine in the LSQE motif were assessed by utilizing specific peptide libraries or individual peptide substrates. All amino acids comprising the LSQE sequence were critical for maximum peptide substrate suitability for ATM. The DNA-dependent protein kinase (DNA-PK), a Ser/Thr kinase related to ATM and important in DNA repair, was compared with ATM in terms of peptide substrate selectivity. DNA-PK was found to be unique in its preference of neighboring amino acids to the phosphorylated serine. Peptide library analyses defined a preferred amino acid motif for ATM that permits clear distinctions between ATM and DNA-PK kinase activity. Data base searches using the library-derived ATM sequence identified previously characterized substrates of ATM, as well as novel candidate substrate targets that may function downstream in ATM-directed signaling pathways. 相似文献
5.
《DNA Repair》2014
Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress. 相似文献
6.
ATM (ataxia-telangiectasia mutated), ATR (ATM- and Rad3-related) and DNA-PK (DNA-dependent protein kinase), important regulators of genome stability, belong to the PIKK (phosphoinositide 3-kinase-like kinase) family of protein kinases. In the present study, DNA-affinity chromatography was used to identify DNA-binding proteins phosphorylated by these kinases. This resulted in the identification of FUS (fused in sarcoma)/TLS (translocated in liposarcoma) as an in vitro target of the PIKKs. FUS is a member of the Ewing's sarcoma family of proteins that appears to play a role in regulating genome stability, since mice lacking FUS show chromosomal instability and defects in meiosis. The residues in FUS that are phosphorylated in vitro and in vivo were identified, and phospho-specific antibodies were generated to demonstrate that FUS becomes phosphorylated at Ser(42) in vivo, primarily in response to agents that cause DSBs (double-strand breaks). DSB-induced FUS phosphorylation in vivo at Ser(42) requires ATM and not DNA-PK. Although Ser(42) is retained in the oncogenic FUS-CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein 10] fusion generated by a t(12;16)(q13;p11) chromosomal translocation, Ser(42) in FUS-CHOP is not phosphorylated after DNA damage. These results identify FUS as a new target of the ATM-signalling pathway and strengthen the notion that FUS regulates genome stability. 相似文献
7.
The human telomerase RNA component,hTR, activates the DNA-dependent protein kinase to phosphorylate heterogeneous nuclear ribonucleoprotein A1
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Nicholas S. Y. Ting Brant Pohorelic Yaping Yu Susan P. Lees-Miller Tara L. Beattie 《Nucleic acids research》2009,37(18):6105-6115
Telomere integrity in human cells is maintained by the dynamic interplay between telomerase, telomere associated proteins, and DNA repair proteins. These interactions are vital to suppress DNA damage responses and unfavorable changes in chromosome dynamics. The DNA-dependent protein kinase (DNA-PK) is critical for this process. Cells deficient for functional DNA-PKcs show increased rates of telomere loss, accompanied by chromosomal fusions and translocations. Treatment of cells with specific DNA-PK kinase inhibitors leads to similar phenotypes. These observations indicate that the kinase activity of DNA-PK is required for its function at telomeres possibly through phosphorylation of essential proteins needed for telomere length maintenance. Here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a direct substrate for DNA-PK in vitro. Phosphorylation of hnRNP A1 is stimulated not only by the presence of DNA but also by the telomerase RNA component, hTR. Furthermore, we show that hnRNP A1 is phosphorylated in vivo in a DNA-PK-dependent manner and that this phosphorylation is greatly reduced in cell lines which lack hTR. These data are the first to report that hTR stimulates the kinase activity of DNA-PK toward a known telomere-associated protein, and may provide further insights into the function of DNA-PK at telomeres. 相似文献
8.
9.
Phosphatidyl inositol 3-kinase-like serine/threonine protein kinases (PIKKs) are required for DNA damage-induced phosphorylation of the 32 kDa subunit of replication protein A at threonine 21 总被引:2,自引:2,他引:2
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Replication protein A (RPA) is a single-stranded DNA (ssDNA) binding protein involved in various processes, including nucleotide excision repair and DNA replication. The 32 kDa subunit of RPA (RPA32) is phosphorylated in response to various DNA-damaging agents, and two protein kinases, ataxia-telangiectasia mutated (ATM) and the DNA-dependent protein kinase (DNA-PK) have been implicated in DNA damage-induced phosphorylation of RPA32. However, the relative roles of ATM and DNA-PK in the site-specific DNA damage-induced phosphorylation of RPA32 have not been reported. Here we generated a phosphospecific antibody that recognizes Thr21-phosphorylated RPA32. We show that both DNA-PK and ATM phosphorylate RPA32 on Thr21 in vitro. Ionizing radiation (IR)-induced phosphorylation of RPA32 on Thr21 was defective in ATM-deficient cells, while camptothecin (CPT)-induced phosphorylation of RPA32 on Thr21 was defective in cells lacking functional DNA-PK. Neither ATM nor DNA-PK was required for etoposide (ETOP)-induced RPA32 Thr21 phosphorylation. However, two inhibitors of the ATM- and Rad3-related (ATR) protein kinase activity prevented ETOP-induced Thr21 phosphorylation. Inhibition of DNA replication prevented both the IR- and CPT-induced phosphorylation of Thr21, whereas ETOP-induced Thr21 phosphorylation did not require active DNA replication. Thus, the regulation of RPA32 Thr21 phosphorylation by multiple DNA damage response protein kinases suggests that Thr21 phosphorylation of RPA32 is a crucial step within the DNA damage response. 相似文献
10.
DNA-dependent protein kinase (DNA-PK) phosphorylates several cellular proteins in vitro, but its cellular function and natural substrate(s) in vivo are not established. We reported activation of DNA ligase in cultured normal human epidermal keratinocytes (NHEK) on exposure to the DNA-damaging compound bis-(2-chloroethyl) sulfide. The activated enzyme was identified as DNA ligase I, and this activation was attributed to phosphorylation of the enzyme. Here, we show that the phosphorylation is mediated by DNA-PK and that DNA ligase I is one of its natural substrates in vivo. DNA ligase I phosphorylation-cum-activation is a response specific to DNA double-strand breaks. We also demonstrate that affinity-purified inactive DNA ligase I is phosphorylated and activated in vitro by HeLa Cell DNA-PK confirming the in vivo observations. The findings specify the roles of DNA-PK and DNA ligase I in mammalian DNA double-strand break repair. 相似文献
11.
Involvement of DNA-dependent protein kinase in regulation of stress-induced JNK activation. 总被引:2,自引:0,他引:2
DNA-dependent protein kinase (DNA-PK) is composed of a 460-kDa catalytic subunit and the regulatory subunits Ku70 and Ku80. The complex is activated on DNA damage and plays an essential role in double-strand-break repair and V(D)J recombination. In addition, DNA-PK is involved in S-phase checkpoint arrest following irradiation, although its role in damage-induced checkpoint arrest is not clear. In an effort to understand the role of DNA-PK in damage signaling, human and mouse cells containing the DNA-PK catalytic subunit (DNA-PKcs proficient) were compared with those lacking DNA-PKcs for c-Jun N-terminal kinase (JNK) activity that mediates physiologic responses to DNA damage. The DNA-PKcs-proficient cells showed much tighter regulation of JNK activity after DNA damage, while the level of JNK protein in both cell lines remained unchanged. The JNK proteins physically associated with DNA-PKcs and Ku70/Ku80 heterodimer, and the interaction was significantly stimulated after DNA damage. Various JNK isoforms not only contained a DNA-PK phosphorylation consensus site (serine followed by glutamine) but also were phosphorylated by DNA-PK in vitro. Together, our results suggest that DNA damage induces physical interaction between DNA-PK and JNK, which may in turn negatively affect JNK activity through JNK phosphorylation by DNA-PK. 相似文献
12.
Beli P Lukashchuk N Wagner SA Weinert BT Olsen JV Baskcomb L Mann M Jackson SP Choudhary C 《Molecular cell》2012,46(2):212-225
The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair. 相似文献
13.
Quanz M Herbette A Sayarath M de Koning L Dubois T Sun JS Dutreix M 《The Journal of biological chemistry》2012,287(12):8803-8815
DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone. 相似文献
14.
DNA-activated protein kinase in Raji Burkitt's lymphoma cells. Phosphorylation of c-Myc oncoprotein.
Autophosphorylation of a DNA-activated protein kinase (DNA-PK) in Raji Burkitt's lymphoma cells generated a band that corresponded to a phosphoprotein of about 300 kDa on SDS/PAGE. This band corresponds to a 300-350-kDa DNA-PK found previously in HeLa cells. In addition to the 300-kDa phosphoprotein, the band of a highly phosphorylated 58-kDa protein was detected by SDS/PAGE of partially purified DNA-PK preparations after the phosphorylation reaction in the presence of double-stranded DNA. This phosphoprotein was specifically immunoprecipitated by phosphoprotein nor detectable activities of other kinases, phosphorylated recombinant c-Myc proteins in the presence of DNA. The c-Myc phosphorylation by DNA-PK was markedly stimulated by relaxed, double-stranded DNA, but neither by single-stranded DNA nor by RNA. Phosphopeptide mapping and phosphoamino acid analysis indicated that DNA-PK phosphorylates c-Myc in vitro at several serine residues. 相似文献
15.
16.
Regulation of CHK2 by DNA-dependent protein kinase 总被引:5,自引:0,他引:5
Chk2 is a critical mediator of diverse cellular responses to DNA damage. Activation of Chk2 by DNA damage requires phosphorylation at sites including Thr68. In earlier work, we found that an activity present in rabbit reticulocyte lysates phosphorylates and activates Chk2. We now find that hypophosphorylated Chk2 can be phosphorylated at Thr68 by various subcellular fractions of HEK293 cells. This activity is sensitive to the phosphatidylinositol 3'-kinase-like kinase inhibitor wortmannin, but not to caffeine. DNA enhances the Chk2 phosphorylation by cellular fractions in vitro. The wortmannin-sensitive Chk2 kinase activity is present in fractions from ATM-deficient cells. In contrast, Chk2 was not efficiently phosphorylated at Thr68 in vitro by fractions from cells with a defective DNA-dependent protein kinase (DNA-PK) catalytic subunit. Chk2 is phosphorylated by purified DNA-PK in vitro. Endogenous Chk2 coimmunoprecipitates Ku70 and Ku80. In a series of matched cell lines having and lacking functional DNA-PK, Chk2 activation by exposure of cells to ionizing radiation, or to camptothecin was consistently diminished in the absence of DNA-PK. Down-regulation of DNA-PK(cs) by either siRNA or a chemical inhibitor attenuated radiation-induced Chk2 phosphorylation. Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage. 相似文献
17.
Vasodilator‐stimulated phosphoprotein (VASP) is a 39‐kDa protein belonging to the Ena/VASP protein family, which is involved in adhesion, migration, cell–cell interaction, and regulation of pathways connected with actin cytoskeleton remodeling. VASP is phosphorylated at Tyr39, Ser157, Ser239, Thr278, and Ser322 mainly by tyrosine kinase Abl, cAMP‐dependent protein kinase, protein kinase G, AMP‐activated protein kinase, and protein kinase D1, respectively. VASP phosphorylation, as a regulator of actin dynamics, may lead to impaired reorganization of the podocyte actin cytoskeleton not only by indirect interaction of VASP with actin but also by regulation of other signaling pathways. A few studies have shown that VASP participates in the development of renal diseases and mediates podocyte movement through its interaction with proteins of the slit diaphragm. VASP phosphorylation may cause reduced actin filament assembly in podocytes and mediate disturbances in regulation of filtration barrier permeability as a consequence of podocyte foot process effacement. In this paper, we describe the role of VASP in podocyte function, mainly in the context of actin dynamics and glomerular filtration barrier permeability. In addition, we discuss the involvement of VASP and its phosphorylated forms in the development of kidney diseases. 相似文献
18.
《Free radical biology & medicine》2006,40(12):1650-1659
DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PKpThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PKpThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs. 相似文献
19.
Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II
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African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an "aster" within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly. 相似文献
20.
Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process. 相似文献