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Artykuły w czasopismach na temat "Ligase IV/XRCC4"

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Nick McElhinny, Stephanie A., Carey M. Snowden, Joseph McCarville i Dale A. Ramsden. "Ku Recruits the XRCC4-Ligase IV Complex to DNA Ends". Molecular and Cellular Biology 20, nr 9 (1.05.2000): 2996–3003. http://dx.doi.org/10.1128/mcb.20.9.2996-3003.2000.

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ABSTRACT Genetic experiments have determined that Ku, XRCC4, and ligase IV are required for repair of double-strand breaks by the end-joining pathway. The last two factors form a tight complex in cells. However, ligase IV is only one of three known mammalian ligases and is intrinsically the least active in intermolecular ligation; thus, the biochemical basis for requiring this ligase has been unclear. We demonstrate here a direct physical interaction between the XRCC4-ligase IV complex and Ku. This interaction is stimulated once Ku binds to DNA ends. Since XRCC4-ligase IV alone has very low DNA binding activity, Ku is required for effective recruitment of this ligase to DNA ends. We further show that this recruitment is critical for efficient end-joining activity in vitro. Preformation of a complex containing Ku and XRCC4-ligase IV increases the initial ligation rate 20-fold, indicating that recruitment of the ligase is an important limiting step in intermolecular ligation. Recruitment by Ku also allows XRCC4-ligase IV to use Ku's high affinity for DNA ends to rapidly locate and ligate ends in an excess of unbroken DNA, a necessity for end joining in cells. These properties are conferred only on ligase IV, because Ku does not similarly interact with the other mammalian ligases. We have therefore defined cell-free conditions that reflect the genetic requirement for ligase IV in cellular end joining and consequently can explain in molecular terms why this factor is required.
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Wu, Peï-Yu, Philippe Frit, SriLakshmi Meesala, Stéphanie Dauvillier, Mauro Modesti, Sara N. Andres, Ying Huang i in. "Structural and Functional Interaction between the Human DNA Repair Proteins DNA Ligase IV and XRCC4". Molecular and Cellular Biology 29, nr 11 (30.03.2009): 3163–72. http://dx.doi.org/10.1128/mcb.01895-08.

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ABSTRACT Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.
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Malashetty, Vidyasagar, Audrey Au, Jose Chavez, Mary Hanna, Jennifer Chu, Jesse Penna i Patricia Cortes. "The DNA binding domain and the C-terminal region of DNA Ligase IV specify its role in V(D)J recombination". PLOS ONE 18, nr 2 (24.02.2023): e0282236. http://dx.doi.org/10.1371/journal.pone.0282236.

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DNA Ligase IV is responsible for the repair of DNA double-strand breaks (DSB), including DSBs that are generated during V(D)J recombination. Like other DNA ligases, Ligase IV contains a catalytic core with three subdomains—the DNA binding (DBD), the nucleotidyltransferase (NTD), and the oligonucleotide/oligosaccharide-fold subdomain (OBD). Ligase IV also has a unique C-terminal region that includes two BRCT domains, a nuclear localization signal sequence and a stretch of amino acid that participate in its interaction with XRCC4. Out of the three mammalian ligases, Ligase IV is the only ligase that participates in and is required for V(D)J recombination. Identification of the minimal domains within DNA Ligase IV that contribute to V(D)J recombination has remained unresolved. The interaction of the Ligase IV DNA binding domain with Artemis, and the interaction of its C-terminal region with XRCC4, suggest that both of these regions that also interact with the Ku70/80 heterodimer are important and might be sufficient for mediating participation of DNA Ligase IV in V(D)J recombination. This hypothesis was investigated by generating chimeric ligase proteins by swapping domains, and testing their ability to rescue V(D)J recombination in Ligase IV-deficient cells. We demonstrate that a fusion protein containing Ligase I NTD and OBDs flanked by DNA Ligase IV DBD and C-terminal region is sufficient to support V(D)J recombination. This chimeric protein, which we named Ligase 37, complemented formation of coding and signal joints. Coding joints generated with Ligase 37 were shorter than those observed with wild type DNA Ligase IV. The shorter length was due to increased nucleotide deletions and decreased nucleotide insertions. Additionally, overexpression of Ligase 37 in a mouse pro-B cell line supported a shift towards shorter coding joints. Our findings demonstrate that the ability of DNA Ligase IV to participate in V(D)J recombination is in large part mediated by its DBD and C-terminal region.
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Mahajan, Kiran N., Stephanie A. Nick McElhinny, Beverly S. Mitchell i Dale A. Ramsden. "Association of DNA Polymerase μ (pol μ) with Ku and Ligase IV: Role for pol μ in End-Joining Double-Strand Break Repair". Molecular and Cellular Biology 22, nr 14 (15.07.2002): 5194–202. http://dx.doi.org/10.1128/mcb.22.14.5194-5202.2002.

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ABSTRACT Mammalian DNA polymerase μ (pol μ) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol μ protein increase. pol μ also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of γH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol μ is thus part of the cellular response to DNA double-strand breaks. pol μ also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol μ in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase β does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol μ in facilitating joining mediated by these factors. Our data thus support an important role for pol μ in the end-joining pathway for repair of double-strand breaks.
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Przewloka, Marcin R., Paige E. Pardington, Steven M. Yannone, David J. Chen i Robert B. Cary. "In Vitro and In Vivo Interactions of DNA Ligase IV with a Subunit of the Condensin Complex". Molecular Biology of the Cell 14, nr 2 (luty 2003): 685–97. http://dx.doi.org/10.1091/mbc.e01-11-0117.

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Several findings have revealed a likely role for DNA ligase IV, and interacting protein XRCC4, in the final steps of mammalian DNA double-strand break repair. Recent evidence suggests that the human DNA ligase IV protein plays a critical role in the maintenance of genomic stability. To identify protein–protein interactions that may shed further light on the molecular mechanisms of DSB repair and the biological roles of human DNA ligase IV, we have used the yeast two-hybrid system in conjunction with traditional biochemical methods. These efforts have resulted in the identification of a physical association between the DNA ligase IV polypeptide and the human condensin subunit known as hCAP-E. The hCAP-E polypeptide, a member of the Structural Maintenance of Chromosomes (SMC) super-family of proteins, coimmunoprecipitates from cell extracts with DNA ligase IV. Immunofluorescence studies reveal colocalization of DNA ligase IV and hCAP-E in the interphase nucleus, whereas mitotic cells display colocalization of both polypeptides on mitotic chromosomes. Strikingly, the XRCC4 protein is excluded from the area of mitotic chromosomes, suggesting the formation of specialized DNA ligase IV complexes subject to cell cycle regulation. We discuss our findings in light of known and hypothesized roles for ligase IV and the condensin complex.
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Francis, Dailia B., Mikhail Kozlov, Jose Chavez, Jennifer Chu, Shruti Malu, Mary Hanna i Patricia Cortes. "DNA Ligase IV regulates XRCC4 nuclear localization". DNA Repair 21 (wrzesień 2014): 36–42. http://dx.doi.org/10.1016/j.dnarep.2014.05.010.

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Roy, Sunetra, Abinadabe J. de Melo, Yao Xu, Satish K. Tadi, Aurélie Négrel, Eric Hendrickson, Mauro Modesti i Katheryn Meek. "XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation". Molecular and Cellular Biology 35, nr 17 (22.06.2015): 3017–28. http://dx.doi.org/10.1128/mcb.01503-14.

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The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA endsin vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNAin vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.
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Recuero-Checa, María A., Andrew S. Doré, Ernesto Arias-Palomo, Angel Rivera-Calzada, Sjors H. W. Scheres, Joseph D. Maman, Laurence H. Pearl i Oscar Llorca. "Electron microscopy of Xrcc4 and the DNA ligase IV–Xrcc4 DNA repair complex". DNA Repair 8, nr 12 (grudzień 2009): 1380–89. http://dx.doi.org/10.1016/j.dnarep.2009.09.007.

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Hayden, Patrick, Prerna Tewari, Anthony Staines, Derek Morris, Dominique Crowley, Alexandra Nieters, Nicholas Becker i in. "Variation in DNA Repair Genes XRCC3, XRCC4, and XRCC5 and Risk of Myeloma." Blood 108, nr 11 (16.11.2006): 3416. http://dx.doi.org/10.1182/blood.v108.11.3416.3416.

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Abstract Aberrant class switch recombination (CSR), the physiological process that regulates maturation of the antibody response, is believed to be an early event in the pathogenesis of myeloma. The genetic basis of CSR, from initiation of the DNA double-strand break through to detection and repair, has been elucidated. We hypothesised that germline polymorphisms in the genes implicated in DNA double strand break repair may contribute to susceptibility to myeloma. We therefore assessed 32 SNPs in 3 genes central to the DNA repair pathway in patients with myeloma and controls from the EpiLymph Study, a European study of the epidemiology of lymphoid neoplasms, and from an Irish hospital registry (306 cases, 263 controls). The genes examined were XRCC3, XRCC4, and XRCC5. XRCC3 is a member of the RecA/Rad51-related protein family that participates in homologous recombination. The XRCC4 protein forms a complex with DNA ligase IV and DNA-dependent protein kinase in the repair of DNA double-strand breaks by non-homologous end joining. XRCC5 encodes the 80-kilodalton subunit of the Ku heterodimer protein, the DNA-binding component of the DNA-dependent protein kinase. SNPs from the chosen genes were identified from HapMap CEU Phase I and II genotype data (Public Release #20; 2006-01-24). Haplotype-tagging SNPs (htSNPs) were chosen based on Tagger analysis (as implemented in Haploview Version 3.2). A SNP in GSTP1 was also genotyped to allow for comparison with allele frequencies previously generated from a myeloma cohort. Genotyping was performed using TaqMan®-based assays on the 7900 ABI HT platform. The drop-out rate was consistently less than 3% in all assays. For quality control (QC) purposes, duplicates of 10% of the samples were interspersed throughout the plates. The concordance rates for QC samples were greater than 98%. GSTP1 SNP results were comparable to previously published findings. For the htSNP rs963248 in XRCC4, Allele A was significantly more frequent in cases than in controls (86.4% vs.80.8%) (p=0.0105), as was the AA genotype (74% vs. 65%) (p=0.026). Haplotype analysis was performed using Cocaphase for rs963248 in combination with additional SNPs in XRCC4. The strongest evidence of association came from the A - T haplotype from rs963248-rs2891980 (80.9% vs. 74.5%; p=0.008). For XRCC5, the genotype GG from rs1051685 was detected in 10 cases from different national populations but in only 1 control (p=0.015). Interestingly, this SNP is located in the 3′ UTR of XRCC5. Overall, these data provide support for the hypothesis that common variation in the genes encoding DNA repair proteins contributes to susceptibility to myeloma.
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Hsu, Hsin-Ling, Steven M. Yannone i David J. Chen. "Defining interactions between DNA-PK and ligase IV/XRCC4". DNA Repair 1, nr 3 (marzec 2002): 225–35. http://dx.doi.org/10.1016/s1568-7864(01)00018-0.

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Rozprawy doktorskie na temat "Ligase IV/XRCC4"

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Stiff, Thomas. "Characterisation of proteins that stimulate the DNA ligase IV/Xrcc4 complex". Thesis, University of Sussex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249402.

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Wang, Yu. "Mismatch ligation during non-homologous end joining pathway kinetic characterization of human DNA ligase IV/XRCC4 complex /". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1179947467.

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De, Melo Abinadabe Jackson. "Molecular basis for the structural role of human DNA ligase IV". Thesis, Aix-Marseille, 2016. http://www.theses.fr/2016AIXM4040.

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Les défauts dans la réparation des cassures double-brin de l'ADN (DSBs) peuvent avoir d'importantes conséquences pouvant entrainer une instabilité génomique et conduire à la mort cellulaire ou au développement de cancers. Dans la plupart des cellules mammifères, le mécanisme de Jonction des Extrémités Non Homologues (NHEJ) est le principal mécanisme de réparation des DSBs. L'ADN Ligase IV (LigIV) est une protéine unique dans sa capacité à promouvoir la NHEJ classique. Elle s'associe avec deux autres protéines structuralement similaires, XRCC4 et XLF (ou Cernunnos). LigIV interagit directement avec XRCC4 pour former un complexe stable, tandis que l'interaction entre XLF et ce complexe est médiée par XRCC4. XLF stimule fortement l'activité de ligation du complexe LigIV/XRCC4 par un mécanisme encore indéterminé. Récemment, un rôle structurel non catalytique a été attribué à LigIV (Cottarel et al., 2013). Dans le travail de thèse présenté ici, nous avons reconstitué l'étape de ligation de la NHEJ en utilisant des protéines recombinantes produites dans des bactéries afin d’une part, d'explorer les bases moléculaires du rôle structural de LigIV, d’autre part de comprendre le mécanisme par lequel XLF stimule le complexe de ligation, et enfin de mieux comprendre comment ces trois protéines coopèrent au cours de la NHEJ. Nos analyses biochimiques suggèrent que XLF via son interaction avec XRCC4 lié à LigIV, pourrait induire un changement conformationnel dans la LigIV. Ce réarrangement de la ligase exposerait son interface de liaison à l'ADN ce qui lui permettrait alors de ponter deux molécules indépendantes d'ADN, une capacité indépendante de l'activité catalytique de LigIV
Failure to repair DNA double-strand breaks (DSBs) may have deleterious consequences inducing genomic instability and even cell death. In most mammalian cells, Non-Homologous End Joining (NHEJ) is a prominent DSB repair pathway. DNA ligase IV (LigIV) is unique in its ability to promote classical NHEJ. It associates with two structurally related proteins called XRCC4 and XLF (aka Cernunnos). LigIV directly interacts with XRCC4 forming a stable complex while the XLF interaction with this complex is mediated by XRCC4. XLF strongly stimulates the ligation activity of the LigIV/XRCC4 complex by an unknown mechanism. Recently, a structural noncatalytic role of LigIV has been uncovered (Cottarel et al., 2013). Here, we have reconstituted the end joining ligation step using recombinant proteins produced in bacteria to explore not only the molecular basis for the structural role of LigIV, but also to understand the mechanism by which XLF stimulates the ligation complex, and how these three proteins work together during NHEJ. Our biochemical analysis suggests that XLF, through interactions with LigIV/XRCC4 complex, could induce a conformational change in LigIV. Rearrangement of the LigIV would expose its DNA binding interface that is able to bridge two independent DNA molecules. This bridging ability is fully independent of LigIV’s catalytic activity. We have mutated this interface in order to attempt to disrupt the newly identified DNA bridging ability. In vitro analysis of this LigIV mutant will be presented as well as a preliminary in vivo analysis
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Ochi, Takashi. "Non-homologous end-joining DNA double strand break repair : structural studies of the DNA ligase IV/XRCC4 complex". Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609793.

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Amram, Jérémy. "Etude structurale et fonctionnelle des complexes multi-protéiques de la voie de réparation NHEJ chez l’homme". Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114822/document.

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La voie de réparation NHEJ (Non-Homologous End-Joining) est une voie majeure de réparation des cassures double-brin chez l’homme. Les protéines de cette voie interagissent et forment des complexes dynamiques dont les mécanismes moléculaires sont encore largement méconnus. Nous avons dans un premier temps mis au point des protocoles de production à l’échelle de plusieurs milligrammes des protéines cœur de la voie NHEJ en cellules d’insecte à l’aide du système MultiBac. Nous avons ainsi purifié les complexes Ku70/Ku80 et Ligase4/XRCC4 et les protéines Cernunnos et Artemis à homogénéité. Des essais de cristallisation, des études par SAXS et des analyses par microscopie électronique ont été réalisés sur différents complexes formés par ces protéines cœur du NHEJ. Nous avons également caractérisé par chromatographie d’exclusion de taille et calorimétrie, les interactions effectuées entre les protéines de la voie NHEJ. L’ensemble de ces travaux a permis d’établir des bases biochimiques solides en vue des études structurales et fonctionnelles de la voie NHEJ chez l’homme
Human DNA repair pathway NHEJ (Non-Homologous End-Joining) is a major pathway of double-strand breaks repair. The proteins involved in this pathway interact and form dynamic complexes whose molecular mechanisms are largely unknown. Firstly, we established protocols to be able to purify milligrams of those NHEJ pathway core proteins using MultiBac insect cells system. We then purified Ku70/Ku80 and Ligase4/XRCC4 complexes, Artemis and Cernunnos to homogeneity. Crystallogenesis assays, SAXS experiments and Transmission Electronic Microscopy experiments have been performed on several complexes formed by these core NHEJ proteins. We also characterized the interactions between these proteins by Size Exclusion Chromatography and Isothermal Calorimetry. These experiments have led to biochemical results sufficient to establish a solid basis to initiate the structural and functional study of the Human NHEJ Pathway
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Menchon, Grégory. "Criblage virtuel et fonctionnel sur le complexe XRCC4/ADN ligase IV/Cer-XLF de ligature des cassures double-brin de l'ADN : application en radiosensibilisation tumorale". Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30395.

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En cancérologie, la radiothérapie est une des armes essentielles pour éradiquer les cellules tumorales. Les cassures des deux brins de l'ADN dites "double-brin" qu'elle induit sont particulièrement toxiques et constituent la principale cause de mort cellulaire. La NHEJ (Jonction d'Extrémités Non-Homologues) est la voie métabolique majeure de réparation de ces cassures double-brin de l'ADN et par ce mécanisme, les cellules humaines adoptent une résistance à la radiothérapie. Ce mécanisme de réparation constitue donc une cible de choix pour un traitement anticancéreux combiné en vue d'augmenter la sensibilité des cellules cancéreuses aux rayons ionisants (radiosensibilisation). Au cours du mécanisme NHEJ, la ligature finale des extrémités d'ADN est assurée par le complexe protéique tripartite: XRCC4/ADN Ligase IV/Cernunnos-XLF. Les interfaces protéiques concernées représentent toutes des cibles potentielles dans une stratégie rationnelle d'isolement de molécules inhibitrices, guidée par les structures tridimensionnelles de chaque protéine. A travers des expériences de criblage virtuel et de validation à la fois biophysique et biochimique, nous avons isolé les premières molécules capable de prévenir in vitro les interactions protéine-protéine pour les complexes XRCC4/Lig4 et XRCC4/Cer-XLF, respectivement. Ces composés sont des points de départ pour l'élaboration d'inhibiteurs potentiels de plus haute affinité grâce à l'apport de la biologie structurale, en vue d'un effet radiosensibilisant cellulaire
Radiotherapy is a major weapon used against cancer. Radio-induced DNA double strand breaks (DSB) are the main lesions responsible for cell death. Non-homologous end-joining (NHEJ) is a predominant DSB repair mechanism which contributes to cancer cells resistance to radiotherapy. NHEJ is thus a good target for strategies which aim at increasing the radio-sensitivity of tumors. Through in silico screening and biophysical and biochemical assays, our objective was to find specific ligands for the XRCC4/Lig4 and XRCC4/Cer-XLF protein-protein interactions involved in NHEJ. Here, we isolated the first compounds able to prevent their interaction in vitro. These early stage inhibitors are promising tools for cancer therapy with the hope to develop more specific compounds for cellular assays through the 3D structure of the protein/inhibitor complexes
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Tatavarthi, Haritha. "Action of Tyrosyl DNA Phosphodiesterase on 3'-Phosphoglycolate Terminated DNA Strand Breaks". VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1799.

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Pandey, Monica. "Understanding the Sequence Dependence of NHEJ Mediated Double-strand Break Repair, and Identification of Novel DNA Ligase Inhibitors and their Potential Use as Cancer Therapeutics". Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4146.

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Repair of DNA breaks is essential for maintenance of genomic integrity. DNA double-strand breaks (DSBs) are considered as the most harmful DNA lesions within the cells which if left unrepaired or are misrepaired, can lead to a wide variety of genetic alterations, chromosomal rearrangements either culminating in oncogenic transformation or apoptosis. Therefore, the cell adopts two major pathways, Homologous recombination (HR) and nonhomologous end joining (NHEJ), in order to repair these DSBs thereby maintaining genome stability. HR mediated DSB repair is error-free and is functional during the S and G2 phases of cell cycle and requires the participation of a sister chromatid to act as a template in the repair process. However, in higher eukaryotes, DSBs are primarily repaired by nonhomologous DNA end joining (NHEJ), which is an error-prone DNA repair mechanism and is active throughout the cell cycle. During NHEJ, KU70/KU80 heterodimer binds to the broken DNA ends and recruits DNA-PKcs, Artemis, and Pol µ or ι to the repair site, resulting in processing of broken DNA ends. Following the end processing, ligation occurs with the help of Ligase IV, XRCC4, XLF and PAXX complex. In general, NHEJ is considered as a DSB repair pathway that does not have a sequence preference and hence thought to be random. Using an oligomer based, cell-free assay system, we evaluated the role of DNA sequence during the end-to-end joining of broken DNA, both at the overhangs and at its flanking regions. Using radiolabeled double-stranded oligomeric DNA substrates, possessing DSBs with different overhang sequences, we observed that there is a distinct difference in joining efficiency of mammalian extracts (rat testis, rat lungs, rat brain and rat heart) when overhangs with G:C rich sequences were studied. In contrast, we observed an enhanced efficiency in DNA end joining when A:T-rich flanking sequences were examined. Specifically, when flanking sequences were homopolymers of A:T or sequences enriched with A:T (adenines and/or thymines), joining efficiency was higher compared to that of random or G:C-rich flanking sequences. In order to investigate the factors that contribute towards the observed sequence specificity of NHEJ, we have overexpressed and purified one of the essential factors involved during NHEJ, the Ligase IV/XRCC4 complex. Ligase IV has a multidomain architecture, consisting of a conserved DNA binding domain at N-terminus and a tandem BRCT domain at C-terminus. The central catalytic domain comprises of adenylation (AD) and oligo-binding (OB-fold) domains. The N-terminal DNA binding domain (DBD) of Ligase IV is crucial for its interaction with DNA. Two hypomorphic mutations (A3V and T9I) in the DBD are observed in Ligase IV syndrome patients. Previous studies suggested that recruitment of Ligase IV/XRCC4 complex is dependent on KU protein complex. Interestingly, we observed that binding of Ligase IV/XRCC4 complex can occur independent of KU70/80, however, in a sequence dependent manner. In the present study, we uncover the sequence preference of Ligase IV/XRCC4 complex for the first time and show that it can bind directly to DNA containing DSBs, when flanked with A:T rich sequence in a KU independent manner irrespective of the sequence of the overhangs. All DNA substrates possessing A:T rich flank sequences investigated in the current study, including the one derived from human genome showed preferential binding to Ligase IV/XRCC4, with a binding constant that was ~10 fold higher than that of G:C rich substrates. Gel mobility shift assays, in conjunction with shift-western blotting assay using various radiolabeled double-stranded oligomers and purified Ligase IV/XRCC4 complex revealed stable DNA-protein complex formation in the absence of KU70/80 heterodimer. Importantly, SCR7, a well-known Ligase IV inhibitor, inhibited the recruitment of Ligase IV/XRCC4 in a concentration dependent manner. The DNA:protein complex formed was resistant to DNase I digestion. In addition, biolayer interferometry (BLI) studies using biotinylated double-stranded oligomers possessing A:T or GC rich sequences flanking the DSBs and purified proteins demonstrated that both Ligase IV/XRCC4 complex and Ligase IV could bind strongly to the immobilized double-stranded oligomer, in a sequence dependent manner (KD values were as low as in nanomolar range for AT rich flanking DNA substrates), unlike XRCC4 (KD= 4.12 ± 0.924 µM). Furthermore, BLI results also revealed that the binding of DNA binding domain (DBD) of Ligase IV was restricted to biotinylated polynucleotide of thymines or AT rich flanking ds DNA substrates. This binding got abrogated when point mutations seen in Ligase IV syndrome patients were introduced to DBD, thereby increasing the KD values many folds. Further, immunodepletion of KU70 protein from mammalian tissue extracts and ex vivo knockdown of Ku70 gene in mammalian cells reduced the joining efficiency of a random DNA substrate, while the NHEJ efficiency of DNA substrates remained unperturbed, when specific sequences were present in the flank region. However, in vitro ligation assays with Ligase IV immunodepleted cell free extracts exhibited reduced NHEJ efficiency irrespective of the substrates used. These observations suggest the unique DNA sequence dependence of Ligase IV involved during classical NHEJ repair pathway, which is KU-independent. The requirement of process of ligation catalysed by any of the three mammalian ligases (Ligase I, III or IV) is absolute, during physiological processes such as DNA replication, recombination and in almost all DNA repair pathways. This makes DNA ligases as attractive therapeutic targets to treat cancer. All three DNA ligases share a high degree of homology both at structural and functional levels (Ligase IV shares 13% and 15% sequence identity to Ligase I and Ligase III, respectively). They possess a well conserved catalytic domain but differ particularly in the DNA recognizing well conserved DNA Binding domain (DBD), that if bound by small molecules, the joining activity of ligases might get severely impaired. Based on homology modelling, and considering the anti-tumor properties of a well-known Ligase IV inhibitor, SCR7, we describe its first water soluble, auto cyclized and oxidized form, known as Sodium salt of SCR7-Pyrazine (Na-SCR7-P). Na-SCR7-P exhibited enhanced bioavailability, unlike the other DMSO-soluble Ligase inhibitors. In the present study, we found that like its parental compound (SCR7), Na-SCR7-P, also inhibited NHEJ in a Ligase IV dependent manner. However, unlike SCR7, it blocked joining catalysed by all three ligases in vitro, making it as an ideal tool for cancer therapeutic studies, as it may target multiple DNA transaction processes within the cancer cells. In depth studies revealed that Na-SCR7-P treatment resulted in reduction of mitochondrial membrane potential and activation of apoptosis culminating in cell death in various cancer cell lines. Importantly, administration of Na-SCR7-P led to significant reduction in tumor growth from 12th day of treatment and its impact was significantly higher than previously described SCR7, which predominantly targets Ligase IV within cells. Antitumor activity of Na-SCR7-P in mice resulted in enhanced lifespan, with minimal side effects. In addition, in ovo chorio-allantoic membrane assay revealed the potent anti-angiogenic property of Na-SCR7-P. Thus, we successfully identified another potent DNA ligase inhibitor, Na-SCR7-P that can potentially be used as a strategy for cancer treatment, owing to its water solubility. In addition, based on various biochemical and biophysical screening approaches, we identified two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Considering the indispensable role of DNA Ligase I during physiological processes such as DNA replication, repair and recombination, DNA Ligase I is considered as an important target for cancer therapy as it can impede proliferation of cancer cells upon treatment with specific small molecule inhibitors. Both the inhibitors blocked the ligation of nicks on DNA catalysed by cell-free extracts or purified Ligase I in a concentration-dependent manner. Docking studies in conjunction with biolayer interferometry and gel shift assays revealed that both SCR17 and SCR21 can bind to Ligase I, particularly to the DNA Binding Domain of Ligase I with KD values in nanomolar range (39 nM ± 8.88 nM and 42 nM ± 12.49 nM, respectively). The inhibitors did not show significant affinities towards DNA Ligase III/XRCC1 and DNA Ligase IV/XRCC4. Further, addition of DNA Ligase I could restore the joining, when the inhibitors were treated with testicular cell-free extracts. Ex vivo studies using multiple assays showed that even though cell death was limited in the presence of inhibitors in cancer cells, their proliferation was certainly compromised. Hence, we identify two promising DNA Ligase I inhibitors, which can be used in biochemical and cellular assays, and could be further modified and optimized to target cancer cells. In summary, the present study describes a sequence specific mechanism of recruitment of Ligase IV/XRCC4 to the broken DNA ends, which will have implications in understanding the sequence preference of NHEJ, in mammalian cells. In addition, we successfully developed novel DNA Ligase I inhibitors (SCR 17 and SCR21) and, a novel, water-soluble inhibitor (Na-SCR7-P) of NHEJ pathway which can further be improved as novel therapeutic strategies for sensitizing cancer cells to DSBs.
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Książki na temat "Ligase IV/XRCC4"

1

Stiff, Thomas. Characterisation of proteins that stimulate the DNA Ligase IV/Xrcc4 complex. 2002.

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