The Experts below are selected from a list of 7596 Experts worldwide ranked by ideXlab platform
William S Dynan - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair Protein complex
Biochemical and Biophysical Research Communications, 2015Co-Authors: Durga Udayakumar, William S DynanAbstract:Abstract Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54 nrb ) cooperates with Ku Protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku Protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku Protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound Protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent Protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair Proteins to stabilize a synaptic pre-ligation complex.
-
identification of the polypyrimidine tract binding Protein associated splicing factor p54 nrb complex as a candidate dna double strand break rejoining factor
Journal of Biological Chemistry, 2005Co-Authors: Catherine L Bladen, Durga Udayakumar, Yoshihiko Takeda, William S DynanAbstract:The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new Protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding Protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding Protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku Protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related Proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules.
-
Subnuclear localization of Ku Protein: functional association with RNA polymerase II elongation sites.
Molecular and Cellular Biology, 2002Co-Authors: William S DynanAbstract:The Ku Protein is part of the DNA-dependent Protein kinase (DNA-PK), which is required for the nonhomologous end-joining pathway of DNA double-strand break (DSB) repair (53, 59). When a break occurs, Ku binds avidly to the DNA ends, translocates inward, and recruits the 470-kDa DNA-PK catalytic subunit (DNA-PKcs) to form an active Protein kinase complex (19, 26, 74). Proteins in this complex cooperate with XRCC4, DNA ligase IV, and other factors to carry out DSB repair (reviewed in reference 22). Ku-deficient cells are sensitive to ionizing radiation and are unable to complete the process of V(D)J recombination, which involves a DSB intermediate. In addition to its well-documented role in DSB repair, Ku appears to have some interaction with the RNA polymerase II (RNAP II) transcription apparatus. Several reports describe the binding of Ku to promoter regions or its ability to regulate transcription of individual genes (reviewed in reference 21 and also see references 11, 25, 31, and 72). Other reports describe interaction of Ku with the general transcription machinery. DNA-PK efficiently phosphorylates RNAP II in vitro (50), and a fraction of Ku resides in RNAP II-containing complexes (41). The importance of these interactions is suggested by the finding that nuclear extracts of Ku-deficient cells exhibit a characteristic transcription defect (70). Transcription of several different promoters is decreased two- to fivefold relative to that of extracts from a matched Ku-containing cell line (70). The defect is entirely at the level of reinitiation and is not seen in assays in which transcription is limited to a single round. Mechanistic studies suggest that the effect is mediated by direct interaction of Ku with transcription Proteins. Neither DNA-PK-dependent Protein phosphorylation nor stable association of Ku with the template seems to be required (70, 71). One hypothesis is that Ku accelerates recycling of limiting transcription factors to the promoter by influencing the higher-order organization of the transcription apparatus (71). For example, recruitment of a preformed complex is inherently more rapid than recruitment of the same Proteins in a stepwise manner. Previous work was performed in vitro with cell extracts in which the native organization of the transcription apparatus had been extensively disrupted. It was important to extend studies in vivo to determine whether Ku is associated with the transcription apparatus under physiological conditions in normally growing cells and whether disruption of this interaction interferes with transcription. Transcription occurs in vivo within discrete structures, or “transcription factories” (15, 51, 75). RNAP II itself exists in dynamic equilibrium between an elongating isoform, RNAP IIO, and an initiating isoform, RNAP IIA, which differ in the phosphorylation states of their large subunits (subunits known as IIo and IIa, respectively) (reviewed in references 16 and 46). Phosphorylation of the RNAP II large subunit occurs cooperatively within a C-terminal domain composed of tandem copies of a heptad repeat, YSPTSPS (10). Early studies of isoform-specific functions suggested a relatively simple model in which phosphorylation was coupled to initiation of transcription (1, 10, 13, 35). More recent work, using phosphospecific antibodies to characterize isoform function in vivo, provides a complex picture involving multiple kinases, coupling of transcription to RNA processing, and dynamic relocalization of RNAP II within the nucleus (8, 33, 45, 55, 65, 69, 75). In the present study, we characterize the association of Ku with two RNAP II populations defined by reactivity with isoform-specific monoclonal antibodies (MAbs). MAb H5 recognizes the IIo subunit when it is phosphorylated at serine 2 of the heptad repeat. This form is present in transcription complexes that have cleared the promoter and are engaged in efficient, processive elongation (14, 34). MAb 8WG16 recognizes the nonphosphorylated IIa subunit, which is present in preinitiation complexes. Studies with model peptides indicate that both MAbs are highly isoform specific (48). Initiating and elongating isoforms of RNAP II interact with different constellations of transcription factors. One of the factors associated with elongating RNAP II is DRB sensitivity-inducing factor (DSIF), isolated originally on the basis of its ability to sensitize a cell-free transcription system to the effects of the elongation inhibitor DRB (66). DSIF is composed of the human homologues of the Spt4 and Spt5 Proteins, which have been studied in several other organisms (4, 28, 30, 32, 39). In Drosophila, the Spt4/5 complex colocalizes with RNAP IIO on polytene chromosomes and associates with transcribed DNA in a chromatin immunoprecipitation (ChIP) assay (4). We show here that Ku associates with RNAP IIO and DSIF, but not with RNAP IIA or other markers of the preinitiation complex. Association with the elongation complex is mediated, in part, by a discrete structural domain of the Ku80 subunit. The isolated domain acts as a dominant-negative mutant, inhibiting transcription in vitro and in vivo. This suggests that the association between Ku and RNAP II is important for maintenance of global transcription levels.
-
Distinct roles for Ku Protein in transcriptional reinitiation and DNA repair.
Journal of Biological Chemistry, 2001Co-Authors: Robin L. Woodard, Juren Huang, William S DynanAbstract:Abstract Transcriptional reinitiation is a distinct phase of the RNA polymerase II transcription cycle. Prior work has shown that reinitiation is deficient in nuclear extracts from Chinese hamster ovary cells lacking the 80-kDa subunit of Ku, a double-strand break repair Protein, and that activity is rescued by expression of the corresponding cDNA. We now show that Ku increases the amount or availability of a soluble factor that is limiting for reinitiation, that the factor increases the number of elongation complexes associated with the template at all times during the reaction, and that the factor itself does not form a tight complex with DNA. The factor may consist of a preformed complex of transcription Proteins that is stabilized by Ku. A Ku mutant, lacking residues 687–728 in the 80-kDa subunit, preferentially suppresses transcription in Ku-containing extracts, suggesting that Ku interacts directly with Proteins required for reinitiation. The Ku mutant functions normally in a DNA end-joining system, indicating that the functions of Ku in transcription and repair are genetically separable. Based on our results, we present a model in which Ku is capable of undergoing a switch between a transcription factor-associated and a repair-active state.
-
Geometry of a complex formed by double strand break repair Proteins at a single DNA end: Recruitment of DNA-PKcs induces inward translocation of Ku Protein
Nucleic Acids Research, 1999Co-Authors: Sunghan Yoo, William S DynanAbstract:Ku Protein and the DNA-dependent Protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku Protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of Proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku Protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku Protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on Protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating Protein conformation under the conditions tested.
Sunghan Yoo - One of the best experts on this subject based on the ideXlab platform.
-
Targeting Ku Protein for sensitizing of breast cancer cells to DNA-damage.
International Journal of Molecular Medicine, 2004Co-Authors: Li Zhang, Sunghan Yoo, Anatoly Dritschilo, Igor Belyaev, Viatcheslav A. SoldatenkovAbstract:Targeting molecular components that are critically involved in the maintenance of genome stability is a promising approach for overcoming intrinsic tumor cell resistance to DNA-damaging treatments. In mammalian cells, the Ku-dependent non-homologous end-joining repair pathway is the predominant process for the repair of double-strand breaks (DSBs) in DNA. Previously, RNA aptamers were selected to efficiently block DNA-binding activity of the Ku Protein in vitro. In the present study, we have tested the efficacy of RNA aptamers against the Ku Protein as molecular sensitizer of MCF-7 breast carcinoma cells to DNA-damage. Toward this end, we established MCF-7 cell sublines stably expressing SC4 aptamer RNAs under the control of the human 7SL small nuclear RNA gene promoter. Vector-transfected (MCF/7SL) cells and cells stably expressing SC4 aptamers (MCF/SC4) were exposed to the anticancer drug etoposide and cellular responses to DNA-damage were evaluated. We found that the presence of RNA aptamers against Ku Protein enhanced etoposide-induced growth inhibition of MCF-7 breast cancer. The SC4 aptamer-mediated sensitization of MCF-7 cells to the anticancer drug is attributable to an increased susceptibility of these cells to apoptosis. The observed effects cannot be accounted for by the differential expression levels of Ku Protein in control and SC4 aptamer-expressing cells, but are rather due to augmented DNA binding-capacity of Ku Protein, as demonstrated in in vitro studies. Thus, RNA aptamers against Ku Protein show potential to sensitize MCF-7 breast carcinoma cells to DNA-damaging agents.
-
Geometry of a complex formed by double strand break repair Proteins at a single DNA end: Recruitment of DNA-PKcs induces inward translocation of Ku Protein
Nucleic Acids Research, 1999Co-Authors: Sunghan Yoo, William S DynanAbstract:Ku Protein and the DNA-dependent Protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells. Ku Protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex. To characterize the arrangement of Proteins in this complex, we developed a set of photocross-linking probes, each with a single free end. We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs. The new probes allow the binding of one molecule of Ku Protein and one molecule of DNA-PKcs in a defined position and orientation. Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end. Characterization of the Ku Protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn. The presence of ATP had no effect on Protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating Protein conformation under the conditions tested.
-
Photocross-linking of an Oriented DNA Repair Complex Ku BOUND AT A SINGLE DNA END
Journal of Biological Chemistry, 1999Co-Authors: Sunghan Yoo, Amy L. Kimzey, William S DynanAbstract:Abstract Ku Protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku Protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku Protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively.
-
Interaction of Ku Protein and DNA-dependent Protein kinase catalytic subunit with nucleic acids
Nucleic Acids Research, 1998Co-Authors: William S Dynan, Sunghan YooAbstract:The Ku Protein-DNA-dependent Protein kinase system is one of the major pathways by which cells of higher eukaryotes respond to double-strand DNA breaks. The components of the system are evolutionarily conserved and homologs are known from a number of organisms. The Ku Protein component binds directly to DNA ends and may help align them for ligation. Binding of Ku Protein to DNA also nucleates formation of an active enzyme complex containing the DNA-dependent Protein kinase catalytic subunit (DNA-PKcs). The interaction between Ku Protein, DNA-PKcs and nucleic acids has been extensively investigated. This review summarizes the results of these biochemical investigations and relates them to recent molecular genetic studies that reveal highly characteristic repair and recombination defects in mutant cells lacking Ku Protein or DNA-PKcs.
-
Characterization of the RNA Binding Properties of Ku Protein
Biochemistry, 1998Co-Authors: Sunghan Yoo, William S DynanAbstract:Ku Protein, a heterodimer of 70 and 83 kDa polypeptides, is the regulatory component of the DNA-dependent Protein kinase (DNA-PK). Ku Protein binds to DNA ends and is essential for DNA double-strand break repair and V(D)J recombination. Although there is some evidence that Ku Protein also binds RNA, its RNA binding properties have not been systematically explored. In the present study, Ku-binding RNAs were identified using systematic evolution of ligands by exponential enrichment (SELEX) technology. These RNAs were assigned to three classes based on common sequence motifs. Most of the selected RNAs bound to Ku Protein with a Kd < or = 2 nM, comparable to the affinity of DNA fragments for Ku Protein under similar conditions. Many of the RNAs inhibited DNA-PK activity by competing with DNA for a common binding site in Ku Protein. None of several RNAs that were tested activated DNA-PK in the absence of DNA. The identification of diverse RNAs that bind avidly to Ku Protein is consistent with the idea that natural RNAs may serve as modulators of DNA-PK activity. Moreover, the RNAs identified in this study may have utility as tools for experimental manipulation of DNA double-strand break repair activity in cells and cell extracts.
Petrovich Pavel Laktionov - One of the best experts on this subject based on the ideXlab platform.
-
Ku Protein as the main cellular target of cell-surface-bound circulating DNA
Expert Opinion on Biological Therapy, 2012Co-Authors: Anna V. Cherepanova, Alexander V. Bushuev, Tatyana G Duzhak, Ivan A. Zaporozhchenko, Valentin V. Vlassov, Petrovich Pavel LaktionovAbstract:Objective: An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects. Materials and methods: The binding of [32P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane–cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target Proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry. Results and conclusions: Both ss- and ds-ODNs form strong ODN–Protein complexes with similar electrophoretic mobilities after incubation with the MC...
-
Ku Protein as the main cellular target of cell-surface-bound circulating DNA.
Expert opinion on biological therapy, 2012Co-Authors: Anna V. Cherepanova, Alexander V. Bushuev, Tatyana G Duzhak, Ivan A. Zaporozhchenko, Valentin V. Vlassov, Petrovich Pavel LaktionovAbstract:An immunomodulatory activity of circulating DNA (cirDNA) is implemented via the interactions of cirDNA with the targets exposed on the cell membrane and/or intracellular targets. The goal of this work was to identify the cellular targets of immunoinhibiting cell-surface-bound cirDNA (csbDNA) using its oligodeoxyribonucleotide (ODN) analogs containing the nucleotide motifs frequently found in csbDNA and displaying the same effects. The binding of [(32)P]-labeled single- and double-stranded ODNs (ss- and ds-ODNs) with membrane-cytosolic (MC) extracts and living human umbilical vein endothelial cells (HUVEC) was studied by electromobility shift assay (EMSA). Complexes of biotinylated ODNs with target Proteins were affinity isolated using streptavidin Sepharose with subsequent SDS-PAGE and identified by MALDI-TOF mass spectrometry. Both ss- and ds-ODNs form strong ODN-Protein complexes with similar electrophoretic mobilities after incubation with the MC extracts of HUVEC either when added extracellularly or lipofected into cells. The ODN-binding Proteins were identified as the DNA-binding components of DNA-dependent Protein kinase (DNA-PK), namely, Ku70 and Ku80 Proteins. Diverse cellular localizations and functions of the Ku Proteins demand further clarification of Ku70/80 role as a mediator of the csbDNA immunoinhibiting effects.
François Strauss - One of the best experts on this subject based on the ideXlab platform.
-
site specific proteolytic cleavage of Ku Protein bound to dna
Proteins, 1993Co-Authors: Sophie Paillard, François StraussAbstract:Ku Protein, a relatively abundant nuclear Protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this Protein in the cell, however, remains to be elucidated. We have observed here that Ku Protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku Protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku Protein, denoted Ku′, has DNA binding properties similar to those of Ku Protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku Protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku Protein function at some stage of the cell cycle. © 1993 Wiley-Liss, Inc.
-
Site‐specific proteolytic cleavage of Ku Protein bound to DNA
Proteins: Structure Function and Genetics, 1993Co-Authors: Sophie Paillard, François StraussAbstract:Ku Protein, a relatively abundant nuclear Protein associated with DNA of mammalian cells, is known to be a heterodimer with subunits of 85 and 72 kDa which binds in vitro to DNA ends and subsequently translocates along the molecule. The functional role played by this Protein in the cell, however, remains to be elucidated. We have observed here that Ku Protein, purified from cultured monkey cells, is the target of specific endoproteolysis in vitro, by which the 85 kDa subunit is cleaved at a precise site while the 72 kDa subunit remains intact. This cleavage releases an 18 kDa polypeptide and converts Ku Protein into a heterodimer composed of the 72 kDa subunit associated with a 69 kDa fragment from the 85 kDa subunit. The proteolyzed form of Ku Protein, denoted Ku′, has DNA binding properties similar to those of Ku Protein. The proteolytic mechanism, which is inhibited by leupeptin and chymostatin, is extremely sensitive to ionic conditions, in particular to pH, being very active at pH 7.0 and completely inhibited at pH 8.0. In addition, cleavage occurs only when Ku Protein is bound to DNA, not free in solution. We suggest that in vivo, such proteolysis might be necessary for Ku Protein function at some stage of the cell cycle. © 1993 Wiley-Liss, Inc.
-
Analysis of the mechanism of interaction of simian Ku Protein with DNA
Nucleic Acids Research, 1991Co-Authors: Sophie Paillard, François StraussAbstract:Ku Protein is a relatively abundant DNA-binding Protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one Protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku Protein with DNA in vitro, using Protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku Protein can bind to hairpin loops and to mononucleosomes. Bound Protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku Protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this Protein in the nucleus.
Nadia Barboule - One of the best experts on this subject based on the ideXlab platform.
-
single stranded dna oligomers stimulate error prone alternative repair of dna double strand breaks through hijacking Ku Protein
Nucleic Acids Research, 2015Co-Authors: Stephen P. Jackson, Ying Yuan, Sebastien Britton, Christine Delteil, Julia Coates, Nadia BarbouleAbstract:In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3′ single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process.
