Ku Antigen Research Articles

The Identification Idea of Antibodies Against Ku and Other High-Frequency Antigens

This study was aimed to provide ideas for identifying the antibodies to high-frequency antigens by analyzing a female case of high-frequency antigen antibody (anti-Ku) using serological and sequencing method. The methods for identification of blood group, erythrocyte antigen, screening and identification of antibody were used to detect the blood type and antibody in the proband. The proband's serum and reagent screening cells treated with Sulfhydryl reagent were applied to judge the type and characteristics of this antibodies when reacted with the regaent screening cells or proband's serum respectively. Gene sequencing was used to determine the genotype of the proband's blood group. The proband's red blood cells were determined as O type RhD positive, whose serum showed strong positive reaction to antibody-screening cells and antibody identification cells with the same intensity in saline and IAT medium, however, the self-cells showed negative effect. The Direct Antihuman Globulin of proband's red blood cells also showed weak positive reaction, and the other blood types were CcEe, Jk(a+b-), P1-, Le(a-b -), Lu (a-b +), K-, k-, Kp(a-b-). Serum of the proband treated with 2-ME still react with three groups of screening cells in IAT medium. The reaction intensity of proband's serum was also unchanged with the cells modified with papain and bromelain, but showed negative effect when the cells were treated with sulfhydryl agents including DTT and 2-ME. Gene sequencing revealed that the KEL genotype of the patient was KEL*02N.24 . This patient had a rare K0 phenotype. The rare Kell-null blood group (also known as K0) were identified by serological and molecular tests in the proband who produced both IgG and IgM type of antibody to high-frequency antigen (anti-Ku). These two methods are of great significance in the identification of this rare blood group as well as the antibody to high frequency antigen.

Self-Penetrating Oligonucleotide Derivatives: Features of Self-Assembly and Interactions with Serum and Intracellular Proteins.

Lipophilic oligonucleotide derivatives are a potent approach to the intracellular delivery of nucleic acids. The binding of these derivatives to serum albumin is a determinant of their fate in the body, as its structure contains several sites of high affinity for hydrophobic compounds. This study focuses on the features of self-association and non-covalent interactions with human serum albumin of novel self-penetrating oligonucleotide derivatives. The study revealed that the introduction of a triazinyl phosphoramidate modification bearing two dodecyl groups at the 3' end region of the oligonucleotide sequence has a negligible effect on its affinity for the complementary sequence. Dynamic light scattering verified that the amphiphilic oligonucleotides under study can self-assemble into micelle-like particles ranging from 8 to 15 nm in size. The oligonucleotides with dodecyl groups form stable complexes with human serum albumin with a dissociation constant of approximately 10-6 M. The oligonucleotide micelles are simultaneously destroyed upon binding to albumin. Using an electrophoretic mobility shift assay and affinity modification, we examined the ability of DNA duplexes containing triazinyl phosphoramidate oligonucleotides to interact with Ku antigen and PARP1, as well as the mutual influence of PARP1 and albumin or Ku antigen and albumin upon interaction with DNA duplexes. These findings, together with the capability of dodecyl-containing derivatives to effectively penetrate different cells, such as HEK293 and T98G, indicate that the oligonucleotides under study can be considered as a platform for the development of therapeutic preparations with a target effect.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

Peptide L1H9 derived from the interaction of structural human rhomboid family 1 and 80-kDa subunit of Ku antigen sensitizes breast cancer to chemoradiotherapy

Peptide L1H9 derived from the interaction of structural human rhomboid family 1 and 80-kDa subunit of Ku antigen sensitizes breast cancer to chemoradiotherapy

HMGB1 signaling phosphorylates Ku70 and impairs DNA damage repair in Alzheimer\u2019s disease pathology

DNA damage is increased in Alzheimer’s disease (AD), while the underlying mechanisms are unknown. Here, we employ comprehensive phosphoproteome analysis, and identify abnormal phosphorylation of 70 kDa subunit of Ku antigen (Ku70) at Ser77/78, which prevents Ku70-DNA interaction, in human AD postmortem brains. The abnormal phosphorylation inhibits accumulation of Ku70 to the foci of DNA double strand break (DSB), impairs DNA damage repair and eventually causes transcriptional repression-induced atypical cell death (TRIAD). Cells under TRIAD necrosis reveal senescence phenotypes. Extracellular high mobility group box 1 (HMGB1) protein, which is released from necrotic or hyper-activated neurons in AD, binds to toll-like receptor 4 (TLR4) of neighboring neurons, and activates protein kinase C alpha (PKCα) that executes Ku70 phosphorylation at Ser77/78. Administration of human monoclonal anti-HMGB1 antibody to post-symptomatic AD model mice decreases neuronal DSBs, suppresses secondary TRIAD necrosis of neurons, prevents escalation of neurodegeneration, and ameliorates cognitive symptoms. TRIAD shares multiple features with senescence. These results discover the HMGB1-Ku70 axis that accounts for the increase of neuronal DNA damage and secondary enhancement of TRIAD, the cell death phenotype of senescence, in AD.

The clinical phenotype of patients positive for antibodies to myositis and myositis-related disorders

Inflammatory myopathies are a clinically diverse group of diseases, in which the detection of particular autoantibodies may facilitate diagnosis, treatment, and prognosis. The aim of this report is to summarize our experience with specific autoantibody testing in patients with inflammatory myopathies. Data were collected over the last decade in the Autoimmune Center of the Sheba Medical Center, a tertiary referral hospital. Data regarding patients' positive for autoantibodies against Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, and PM-Scl antigens were retrospectively collected. Patient demographics, clinical characteristics, and mortality were recorded. Descriptive statistics (mean, standard deviation, frequency, and percentage) were calculated. A total of 507 patients were surveyed for sclero-poly-synthetase antibodies, as part of the diagnostic workup of myositis/myalgia or interstitial lung disease. Forty-three patients were found positive for one or more of the abovementioned antibodies, and 23 of them (53.49%) had interstitial lung disease (ILD). Four patients were positive for anti-PL-7, three of them had ILD and Raynaud's phenomenon. Five patients were positive for anti-Ku, and four of them had both arthritis and Raynaud's phenomenon. Nine patients were positive for anti-Mi-2, and six of them were given diagnosed with dermatomyositis. Ten patients were positive for anti-SRP, and six of them had cancers of various types. Our results reiterate the previously recognized associations between anti-Mi-2 and dermatomyositis, anti-Ku and Raynaud's phenomenon, and between anti-PL-7 and ILD. In addition, our data support an association between anti-SRP autoantibody positivity and malignancy, which calls for further investigation.

Ku antigen displays the AP lyase activity on a certain type of duplex DNA

In the search for proteins reactive to apurinic/apyrimidinic (AP) sites, it has been earlier found that proteins of human cell extracts formed the Schiff-base-dependent covalent adduct with an apparent molecular mass of 100kDa with a partial DNA duplex containing an AP site and 5′- and 3′-protruding ends (DDE-AP DNA). The adduct of such electrophoretic mobility was characteristic of only DDE-AP DNA (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777–1785). The protein in this unusual adduct was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF MS data (Kosova et al., Biopolym. Cell 30 (2014) 42–46). Here we studied the interaction of Ku with DDE-AP DNA in details. Purified Ku (the Ku80 subunit) was shown to form the 100-kDa adduct highly specific for AP DNA with a certain length of protruding ends, base opposite the AP site and AP site location. Ku is capable of AP site cleavage in DDE-AP DNA unlike in analogous AP DNA with blunt ends. Ku cleaves AP sites via β-elimination and prefers apurinic sites over apyrimidinic ones. The AP site in DDE-DNA can be repaired in an apurinic/apyrimidinic endonuclease-independent manner via the successive action of Ku (cleavage of the AP site), tyrosyl-DNA phosphodiesterase 1 (removal of the 3ʹ-deoxyribose residue), polynucleotide kinase 3ʹ-phosphatase (removal of the 3ʹ-phosphate), DNA polymerase β (incorporation of dNMP), and DNA ligase (sealing the nick). These results provide a new insight into the role of Ku in the repair of AP sites.

Role of Ku antigen in the repair of apurinic/apyrimidinic sites in DNA

Apurinic/apyrimidinic (AP) sites are some of the most frequent lesions in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku)--the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair--was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here, Ku roles in NHEJ and BER pathways of DNA repair are overviewed.

Ku Antigen Interaction with Apurinic/Apyrimidinic Sites: Nonhomologous End Joining Vs Base Excision Repair

The apurinic/apyrimidinic (AP) site is considered to be a common lesion in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku) - the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair - was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here we make an overview of Ku roles in the NHEJ and BER pathways of DNA repair.

7B7: a novel antibody directed against the Ku70/Ku80 heterodimer blocks invasion in pancreatic and lung cancer cells.

Development of more effective therapeutic strategies for cancers of high unmet need requires the continued discovery of disease-specific protein targets for therapeutic antibody targeting. In order to identify novel proteins associated with cancer cell invasion/metastasis, we present here an alternative to antibody targeting of cell surface proteins with an established role in invasion; our functional antibody screening approach involves the isolation and selection of MAbs that are primarily screened for their ability to inhibit tumour invasion. A clonal population of the Mia PaCa-2, a pancreatic ductal adenocarcinoma (PDAC) cell line, which displays a highly invasive phenotype, was used to generate MAbs with the objective of identifying membrane targets directly involved in cancer invasion. Selected MAb 7B7 can significantly reduce invasion in a dose-responsive manner in Mia PaCa-2 clone 3 and DLKP-M squamous lung carcinoma cells. Using immunoprecipitation and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis, the target antigen of anti-invasive antibody, 7B7, was determined to be the heterodimeric Ku antigen, Ku70/80, a core protein composed of the Ku70 and Ku80 subunits which is involved in non-homologous end-joining (NHEJ) DNA repair. RNA interference-mediated knockdown of Ku70 and Ku80 resulted in a marked decrease in the invasive capacity of Mia PaCa-2 clone 3 and DLKP-M cells, indicating that Ku70/Ku80 is functionally involved in pancreatic and lung cancer invasion. Immunohistochemical analysis demonstrated Ku70/Ku80 immunoreactivity in 37 PDAC tumours, indicating that this heterodimer is highly expressed in this aggressive cancer type. This study demonstrates that a functional MAb screening approach coupled with immunoprecipitation/proteomic analyses can be successfully applied to identify functional anti-invasive MAbs and potential novel targets for therapeutic antibody targeting.

Prévalence et valeur diagnostique des anticorps antinucléaires de spécificité antigénique indéterminée : étude rétrospective à propos d’une série de 90 patients

PurposeThe objective of this study was to determine the clinical relevance and the diagnostic significance of positive antinuclear antibodies (ANA) without identified antigenic target by the usual characterization technique. Patients and methodsRetrospective study conducted in the Laboratory of Immunology of Habib Bourguiba Hospital (Sfax, Tunisia) during 18months. The inclusion criteria were the presence of an ANA titer greater or equal to 1/320 with negative characterization result. ANA screening was performed by indirect immunofluorescence (IIF) on Hep2 cells. Each positive serum was tested by IIF on Crithidia luciliae (anti-native DNA) and by immunodot (anti-nucleosome, anti-histone, anti-Sm, anti-RNP, anti-SSA, anti-SSB, anti-Scl 70, anti-PM-Scl, anti-Jo1, anti-PCNA and anti-ribosomal protein). Sera of systemic lupus erythematosus (SLE), myositis, and scleroderma patients were tested for anti-Ku, anti-PL7, anti-PL12 and anti-Ro-52 using dot myositis. ResultsSera of 90 patients were studied: 18 men and 72 women (average age: 44years). Drug-induced ANA was found in eight patients. The most frequent clinical symptoms were joint (56.7%), cutaneous (54.4%) and constitutional symptoms (45.6%). The diagnosis of an autoimmune disease was suspected in 49 patients (54.5%) and confirmed in 30 (33.3%) including 20 cases of connective tissue disease: myositis (n=6), scleroderma (n=5), Sjögren's syndrome (n=3), SLE (n=4), rheumatoid arthritis (n=6) and antiphospholipid syndrome (n=4). Other autoimmune diseases were less frequent. The anti-Ku antibody was detected in the majority of patients with connective tissue disease. The diagnosis of non-autoimmune diseases was established in 25.5% of patients. Eighteen patients (20%) had no diagnosis orientation. ConclusionOur study demonstrated the diagnostic value of the presence of ANA even in the absence of known antigenic target, confirmed the role of the IIF as “gold standard” test for ANA screening, and suggested the usefulness of the addition of Ku antigen in the immunodot classic profile.

Diagnostic performance and validation of autoantibody testing in myositis by a commercial line blot assay

Serological testing for myositis-specific or associated autoantibodies [myositis-specific antibody (MSA) and myositis-associated antibody (MAA)] is useful for the diagnosis of idiopathic inflammatory myopathies (IIMs). However, available assays are neither standardized nor validated. The objective is to evaluate the accuracy of a commercial line blot assay for myositis diagnosis. IgG antibodies against Jo-1, PL-7, PL-12, PM/Scl, Ku, Mi-2 and Ro52 antigens were detected by a line blot and in-house RNA immunoprecipitation or immunoblot. We tested sera from 208 IIM patients, 50 healthy subjects and 180 control patients (11 non-autoimmune myopathy, 23 muscular dystrophy, 11 UCTD, 68 SLE, 36 SSc, 22 SS and 9 arthropathy). MSAs or MAAs were detected in 98 (47%) out of the 208 IIM patients by line blot: anti-Jo-1 in 43 (21%), anti-PL-7 or anti-PL-12 in 8 (4%), anti-Mi-2 in 9 (4%), anti-PM/Scl in 9 (4%), anti-Ku in 10 (5%) and anti-Ro52 in 49 (24%). Overall specificity was: 100% for anti-Jo-1, anti-PL-7 or PL-12 and anti-PM/Scl; 96% for anti-Ku; 98% for anti-Mi-2; and 76% for anti-Ro52. In-house testing confirmed line blot results regarding anti-Jo-1, anti-PM/Scl and anti-Ku, while it was more accurate than line blot in detecting anti-Mi-2 (7 vs 4% sensitivity, 100 vs 98% specificity), and anti-aminoacyl-tRNA synthetase (anti-ARS) non-Jo-1 antibodies (11 vs 4% sensitivity, 97 vs 99% specificity). Line blot could be a suitable serological test in the diagnostic workup for myositis, and it represents a reliable alternative to more time-consuming procedures. Continuous effort is recommended in order to improve its accuracy.

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku antigen and PP5 for PARP-1 activity subsequent to DSBs.

Tracking Ku antigen levels in cell extracts with DNA containing abasic sites

Prominent lesions in DNA are abasic (AP) sites arising spontaneously or as intermediates during base excision repair. An AP site can form a Schiff base intermediate with primary amino groups of proteins. This intermediate can be stabilized by NaBH 4 treatment and, therefore, cross-linking of AP site-containing DNA (AP DNA) can be used as a tool in detecting proteins that interact with AP sites. Using AP DNA, we observed in the extracts derived from several human cell lines a predominant cross-linked product with an apparent molecular mass of 95 kDa. The cross-linked protein was identified as the p80 subunit of Ku antigen (Ku80) (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777–1785 [1]). Because the cross-linking of Ku80 to AP sites is efficient and selective, this approach may be useful to estimate the amount of Ku antigen in cell extracts in the presence of other cellular proteins. We compared levels of Ku80 detected by dot-ELISA with Ku80 antibodies to the levels of Ku80 cross-linked to AP DNA in extracts derived from HeLa cells and several melanoma cell lines. The level of Ku80 trapping varied considerably depending on the cell lines and correlated with the amount of Ku80 in the extracts estimated by the immunochemical approach. This approach, unlike western blot or estimation of the Ku content based on mRNA levels, is more suitable for tracking Ku forms active in DNA binding including those having aberrations in Ku80, but retaining an ability to heterodimerize with Ku70, that provides efficient loading of Ku antigen onto DNA ends. As a routine test, borohydride trapping (BHT) is also less time and reagent consuming than blotting and EMSA.

Identification of Ku80 subunit of Ku antigen as a protein reactive to apurinic/apyrimidinic sites

Identification of Ku80 subunit of Ku antigen as a protein reactive to apurinic/apyrimidinic sites

Response of heterogeneous ribonuclear proteins (hnRNP) to ionising radiation and their involvement in DNA damage repair

Purpose: To determine the relationship between heterogeneous nuclear ribonucleoproteins (hnRNP) and DNA repair, particularly in response to ionising radiation (IR).Materials and methods: The literature was examined for papers related to the topics of hnRNP, IR and DNA repair.Results: HnRNP orchestrate the processing of mRNA to which they are bound in response to IR. HnRNP A18, B1, C1/C2 and K interact with important proteins from DNA Damage Response (DDR) pathways, binding DNA-dependent protein kinase (DNA-PK), the Ku antigen (Ku) and tumour suppressor protein 53 (p53) respectively. Notably, irregularities in the expression of hnRNP A18, B1, K, P2 and L have been linked to cancer and radiosensitivity. Sixteen different hnRNP proteins have been reported to show either mRNA transcript or protein quantity changes following IR. Various protein modifications of hnRNP in response to IR have also been noted: hnRNP A18, C1/C2 and K are phosphorylated; hnRNP C1/C2 is a target of apoptotic proteases; and hnRNP K degradation is controlled by murine double minute ubiquitin ligase (MDM2). Evidence points to a role for hnRNP A1, A18, A2/B1, C1/C2, K and P2 in regulating double-stranded break (DSB) repair pathways by promoting either homologous recombination (HR) or non-homologous end rejoining (NHEJ) repair pathways following IR.Conclusions: HnRNP proteins play a pivotal role in coordinating repair pathways following exposure to IR, through protein-protein interactions and transcript regulation of key repair and stress response mRNA. In particular, several hnRNP proteins are critical in coordinating the choice of HR or NHEJ to repair DSB caused by IR.

In Vivo Analysis of Protein Kinase B (PKB)/Akt Regulation in DNA-PKcs-null Mice Reveals a Role for PKB/Akt in DNA Damage Response and Tumorigenesis

Full activation of protein kinase B (PKB/Akt) requires phosphorylation on Thr-308 and Ser-473. It is well established that Thr-308 is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1). Ser-473 phosphorylation is mediated by both mammalian target of rapamycin-rictor complex (mTORC2) and DNA-dependent protein kinase (DNA-PK) depending on type of stimulus. However, the physiological role of DNA-PK in the regulation of PKB phosphorylation remains to be established. To address this, we analyzed basal, insulin-induced, and DNA damage-induced PKB Ser-473 phosphorylation in DNA-PK catalytic subunit-null DNA-PKcs(-/-) mice. Our results revealed that DNA-PK is required for DNA damage-induced phosphorylation but dispensable for insulin- and growth factor-induced PKB Ser-473 phosphorylation. Moreover, DNA-PKcs(-/-) mice showed a tissue-specific increase in basal PKB phosphorylation. In particular, persistent PKB hyperactivity in the thymus apparently contributed to spontaneous lymphomagenesis in DNA-PKcs(-/-) mice. Significantly, these tumors could be prevented by deletion of PKBalpha. These findings reveal stimulus-specific regulation of PKB activation by specific upstream kinases and provide genetic evidence of PKB deregulation in DNA-PKcs(-/-) mice.

Expression of apolipoprotein C-IV is regulated by Ku antigen/peroxisome proliferator-activated receptor γ complex and correlates with liver steatosis

We previously reported that hepatitis C virus (HCV) core protein up regulated transcription of apolipoprotein C-IV (ApoC-IV, 10.7-fold increase), a member of the apolipoprotein family implicated in liver steatosis. Here, we identified host transcription factors regulating the ApoC-IV gene expression. Transcriptional regulators were identified by DNA affinity purification and steatosis was detected by oil red O staining and triglyceride assay. We defined a 163-bp ApoC-IV promoter as a core protein responsive element, and identified Ku antigen complex (Ku70 and Ku80) as well as nuclear receptors PPARgamma/RXRalpha as key regulators of ApoC-IV gene expression. Both Ku70 overexpression and PPARgamma agonist significantly increased ApoC-IV promoter activity; conversely, Ku70 silencing or mutation of PPARgamma binding site diminished the ApoC-IV promoter activity. Interestingly, transient transfection of ApoC-IV cDNA into a human hepatoma cell line was able to trigger moderate lipid accumulation. In agreement with this in vitro study, ApoC-IV transcript level was increased in HCV infected livers which correlated with triglyceride accumulation. ApoC-IV overexpression may perturb lipid metabolism leading to lipid accumulation. HCV core protein may modulate ApoC-IV expression through Ku antigen and PPARgamma/RXRalpha complex.

Ku antigen interacts with abasic sites

One of the most abundant lesions in DNA is the abasic (AP) sites arising spontaneously or as an intermediate in base excision repair. Certain proteins participating in the processing of these lesions form a Schiff base with the deoxyribose of the AP site. This intermediate can be stabilized by NaBH 4 treatment. By this method, DNA duplexes with AP sites were used to trap proteins in cell extracts. In HeLa cell extract, along with a prevalent trap product with an apparent molecular mass of 95 kDa, less intensive low-molecular-weight products were observed. The major one was identified as the p80-subunit of Ku antigen (Ku). Ku antigen, a DNA binding component of DNA-dependent protein kinase (DNA-PK), participates in double-stranded break repair and is responsible for the resistance of cells to ionizing radiation. The specificity of Ku interaction with AP sites was proven by more efficient competition of DNA duplexes with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with different efficiencies depending on the size and position of strand interruptions opposite to AP sites. Ku antigen as a part of DNA-PK was shown to inhibit AP site cleavage by apurinic/apyrimidinic endonuclease 1.

Prevalence and clinical associations of anti-Ku antibodies in patients with systemic sclerosis: a European EUSTAR-initiated multi-centre case–control study

Objectives:To determine the prevalence of anti-Ku antibodies in 625 patients with systemic sclerosis (SSc) from six European rheumatological centres and to evaluate their clinical and serological characteristics.Methods:Sera of 625 consecutive...