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Sample records for dna ligase iv

  1. DNA ligase IV syndrome; a review.

    PubMed

    Altmann, Thomas; Gennery, Andrew R

    2016-10-07

    DNA ligase IV deficiency is a rare primary immunodeficiency, LIG4 syndrome, often associated with other systemic features. DNA ligase IV is part of the non-homologous end joining mechanism, required to repair DNA double stranded breaks. Ubiquitously expressed, it is required to prevent mutagenesis and apoptosis, which can result from DNA double strand breakage caused by intracellular events such as DNA replication and meiosis or extracellular events including damage by reactive oxygen species and ionising radiation.Within developing lymphocytes, DNA ligase IV is required to repair programmed DNA double stranded breaks induced during lymphocyte receptor development.Patients with hypomorphic mutations in LIG4 present with a range of phenotypes, from normal to severe combined immunodeficiency. All, however, manifest sensitivity to ionising radiation. Commonly associated features include primordial growth failure with severe microcephaly and a spectrum of learning difficulties, marrow hypoplasia and a predisposition to lymphoid malignancy. Diagnostic investigations include immunophenotyping, and testing for radiosensitivity. Some patients present with microcephaly as a predominant feature, but seemingly normal immunity. Treatment is mainly supportive, although haematopoietic stem cell transplantation has been used in a few cases.

  2. DNA Ligase IV regulates XRCC4 nuclear localization.

    PubMed

    Francis, Dailia B; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-09-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. DNA Ligase IV regulates XRCC4 nuclear localization

    PubMed Central

    Francis, Dailia B.; Kozlov, Mikhail; Chavez, Jose; Chu, Jennifer; Malu, Shruti; Hanna, Mary; Cortes, Patricia

    2014-01-01

    DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620 to 800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4. PMID:24984242

  4. Identification of a novel motif in DNA ligases exemplified by DNA ligase IV.

    PubMed

    Marchetti, Caterina; Walker, Sarah A; Odreman, Federico; Vindigni, Alessandro; Doherty, Aidan J; Jeggo, Penny

    2006-07-13

    DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.

  5. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  6. Defining interactions between DNA-PK and ligase IV/XRCC4

    SciTech Connect

    Hsu, Hsin-Ling; Yannone, Steven M.; Chen, David J.

    2001-04-10

    Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.

  7. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    PubMed

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  8. Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV.

    PubMed

    Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-11-17

    Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia-reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair.

  9. Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV

    PubMed Central

    Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-01-01

    Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia–reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair. PMID:27853172

  10. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    SciTech Connect

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    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.

  11. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    PubMed

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    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.

  12. SCR7 is neither a selective nor a potent inhibitor of human DNA ligase IV.

    PubMed

    Greco, George E; Matsumoto, Yoshihiro; Brooks, Rhys C; Lu, Zhengfei; Lieber, Michael R; Tomkinson, Alan E

    2016-07-01

    DNA ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks.

    PubMed

    Liang, Li; Deng, Li; Nguyen, Son C; Zhao, Xin; Maulion, Christopher D; Shao, Changshun; Tischfield, Jay A

    2008-06-01

    DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.

  14. C-Terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin.

    PubMed

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  15. Molecular and immunological characterization of DNA ligase IV deficiency.

    PubMed

    Jiang, Jinqiu; Tang, Wenjing; An, Yunfei; Tang, Maozhi; Wu, Junfeng; Qin, Tao; Zhao, Xiaodong

    2016-02-01

    DNA ligase IV (LIG4) deficiency is an extremely rare autosomal recessive primary immunodeficiency disease caused by the LIG4 mutation. To date, fewer than 30 cases of patients have been reported worldwide. No reversion mutations have been previously identified in LIG4. This study enrolled seven Chinese patients with LIG4 deficiency who presented with combined immunodeficiency, microcephaly, and growth retardation. One patient (P1) acquired non-Hodgkin lymphoma. Four patients had impaired T cell proliferation function and skewed T cell receptor diversity. Five novel mutations in LIG4 and a potential hotspot mutation (c.833G>T; p.R278L) in the Chinese population were identified. TA cloning analysis of T cells, NK cells, granulocytes, and oral mucosa cells in P6 revealed wild-type clones and clones that contained both maternally and paternally inherited mutations, indicating possible somatic reversion which need further investigation since no functional or protein assays were possible for all the patients died and no cell lines were available.

  16. Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication.

    PubMed

    Muylaert, Isabella; Elias, Per

    2007-04-13

    Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.

  17. DNA ligases.

    PubMed

    Tabor, S

    2001-05-01

    DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

  18. Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule.

    PubMed

    Kysela, Boris; Doherty, Aidan J; Chovanec, Miroslav; Stiff, Thomas; Ameer-Beg, Simon M; Vojnovic, Borivoj; Girard, Pierre-Marie; Jeggo, Penny A

    2003-06-20

    The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

  19. DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells.

    PubMed

    Oh, Sehyun; Harvey, Adam; Zimbric, Jacob; Wang, Yongbao; Nguyen, Thanh; Jackson, Pauline J; Hendrickson, Eric A

    2014-09-01

    Ku-dependent C-NHEJ (classic non-homologous end joining) is the primary DNA EJing (end joining) repair pathway in mammals. Recently, an additional EJing repair pathway (A-NHEJ; alternative-NHEJ) has been described. Currently, the mechanism of A-NHEJ is obscure although a dependency on LIGIII (DNA ligase III) is often implicated. To test the requirement for LIGIII in A-NHEJ we constructed a LIGIII conditionally-null human cell line using gene targeting. Nuclear EJing activity appeared unaffected by a deficiency in LIGIII as, surprisingly, so were random gene targeting integration events. In contrast, LIGIII was required for mitochondrial function and this defined the gene's essential activity. Human Ku:LIGIII and Ku:LIGIV (DNA ligase IV) double knockout cell lines, however, demonstrated that LIGIII is required for the enhanced A-NHEJ activity that is observed in Ku-deficient cells. Most unexpectedly, however, the majority of EJing events remained LIGIV-dependent. In conclusion, although human LIGIII has an essential function in mitochondrial maintenance, it is dispensable for most types of nuclear DSB repair, except for the A-NHEJ events that are normally suppressed by Ku. Moreover, we describe that a robust Ku-independent, LIGIV-dependent repair pathway exists in human somatic cells.

  20. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    PubMed

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

  1. DNA ligase IV as a new molecular target for temozolomide

    SciTech Connect

    Kondo, Natsuko; Takahashi, Akihisa; Mori, Eiichiro; Ohnishi, Ken; McKinnon, Peter J.; Sakaki, Toshisuke; Nakase, Hiroyuki; Ohnishi, Takeo

    2009-10-02

    Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.

  2. DNA damage induced by boron neutron capture therapy is partially repaired by DNA ligase IV.

    PubMed

    Kondo, Natsuko; Sakurai, Yoshinori; Hirota, Yuki; Tanaka, Hiroki; Watanabe, Tsubasa; Nakagawa, Yosuke; Narabayashi, Masaru; Kinashi, Yuko; Miyatake, Shin-ichi; Hasegawa, Masatoshi; Suzuki, Minoru; Masunaga, Shin-ichiro; Ohnishi, Takeo; Ono, Koji

    2016-03-01

    Boron neutron capture therapy (BNCT) is a particle radiation therapy that involves the use of a thermal or epithermal neutron beam in combination with a boron ((10)B)-containing compound that specifically accumulates in tumor. (10)B captures neutrons and the resultant fission reaction produces an alpha ((4)He) particle and a recoiled lithium nucleus ((7)Li). These particles have the characteristics of high linear energy transfer (LET) radiation and therefore have marked biological effects. High-LET radiation is a potent inducer of DNA damage, specifically of DNA double-strand breaks (DSBs). The aim of the present study was to clarify the role of DNA ligase IV, a key player in the non-homologous end-joining repair pathway, in the repair of BNCT-induced DSBs. We analyzed the cellular sensitivity of the mouse embryonic fibroblast cell lines Lig4-/- p53-/- and Lig4+/+ p53-/- to irradiation using a thermal neutron beam in the presence or absence of (10)B-para-boronophenylalanine (BPA). The Lig4-/- p53-/- cell line had a higher sensitivity than the Lig4+/+ p53-/-cell line to irradiation with the beam alone or the beam in combination with BPA. In BNCT (with BPA), both cell lines exhibited a reduction of the 50 % survival dose (D 50) by a factor of 1.4 compared with gamma-ray and neutron mixed beam (without BPA). Although it was found that (10)B uptake was higher in the Lig4+/+ p53-/- than in the Lig4-/- p53-/- cell line, the latter showed higher sensitivity than the former, even when compared at an equivalent (10)B concentration. These results indicate that BNCT-induced DNA damage is partially repaired using DNA ligase IV.

  3. Structural insights into the role of domain flexibility in human DNA ligase IV.

    PubMed

    Ochi, Takashi; Wu, Qian; Chirgadze, Dimitri Y; Grossmann, J Günter; Bolanos-Garcia, Victor M; Blundell, Tom L

    2012-07-03

    Knowledge of the architecture of DNA ligase IV (LigIV) and interactions with XRCC4 and XLF-Cernunnos is necessary for understanding its role in the ligation of double-strand breaks during nonhomologous end joining. Here we report the structure of a subdomain of the nucleotidyltrasferase domain of human LigIV and provide insights into the residues associated with LIG4 syndrome. We use this structural information together with the known structures of the BRCT/XRCC4 complex and those of LigIV orthologs to interpret small-angle X-ray scattering of LigIV in complex with XRCC4 and size exclusion chromatography of LigIV, XRCC4, and XLF-Cernunnos. Our results suggest that the flexibility of the catalytic region is limited in a manner that affects the formation of the LigIV/XRCC4/XLF-Cernunnos complex.

  4. Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.

    PubMed

    Costantini, Silvia; Woodbine, Lisa; Andreoli, Lucia; Jeggo, Penny A; Vindigni, Alessandro

    2007-06-01

    DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.

  5. Structural basis of DNA ligase IV-Artemis interaction in nonhomologous end-joining.

    PubMed

    De Ioannes, Pablo; Malu, Shruti; Cortes, Patricia; Aggarwal, Aneel K

    2012-12-27

    DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

    PubMed

    Chiruvella, Kishore K; Liang, Zhuobin; Birkeland, Shanda R; Basrur, Venkatesha; Wilson, Thomas E

    2013-06-01

    DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

  7. Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs.

    PubMed

    Malu, Shruti; De Ioannes, Pablo; Kozlov, Mikhail; Greene, Marsha; Francis, Dailia; Hanna, Mary; Pena, Jesse; Escalante, Carlos R; Kurosawa, Aya; Erdjument-Bromage, Hediye; Tempst, Paul; Adachi, Noritaka; Vezzoni, Paolo; Villa, Anna; Aggarwal, Aneel K; Cortes, Patricia

    2012-05-07

    Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis-Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA-protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs.

  8. Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs

    PubMed Central

    Malu, Shruti; De Ioannes, Pablo; Kozlov, Mikhail; Greene, Marsha; Francis, Dailia; Hanna, Mary; Pena, Jesse; Escalante, Carlos R.; Kurosawa, Aya; Erdjument-Bromage, Hediye; Tempst, Paul; Adachi, Noritaka; Vezzoni, Paolo; Villa, Anna; Aggarwal, Aneel K.

    2012-01-01

    Artemis is an endonuclease that opens coding hairpin ends during V(D)J recombination and has critical roles in postirradiation cell survival. A direct role for the C-terminal region of Artemis in V(D)J recombination has not been defined, despite the presence of immunodeficiency and lymphoma development in patients with deletions in this region. Here, we report that the Artemis C-terminal region directly interacts with the DNA-binding domain of Ligase IV, a DNA Ligase which plays essential roles in DNA repair and V(D)J recombination. The Artemis–Ligase IV interaction is specific and occurs independently of the presence of DNA and DNA–protein kinase catalytic subunit (DNA-PKcs), another protein known to interact with the Artemis C-terminal region. Point mutations in Artemis that disrupt its interaction with Ligase IV or DNA-PKcs reduce V(D)J recombination, and Artemis mutations that affect interactions with Ligase IV and DNA-PKcs show additive detrimental effects on coding joint formation. Signal joint formation remains unaffected. Our data reveal that the C-terminal region of Artemis influences V(D)J recombination through its interaction with both Ligase IV and DNA-PKcs. PMID:22529269

  9. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    SciTech Connect

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  10. Effects of DNA end configuration on XRCC4-DNA ligase IV and its stimulation of Artemis activity.

    PubMed

    Gerodimos, Christina A; Chang, Howard H Y; Watanabe, Go; Lieber, Michael R

    2017-08-25

    In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases, and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5' and 3' overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3' overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3' overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5' overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3' overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Altered regulation of DNA ligase IV activity by aberrant promoter DNA methylation and gene amplification in colorectal cancer.

    PubMed

    Kuhmann, Christine; Li, Carmen; Kloor, Matthias; Salou, Mariam; Weigel, Christoph; Schmidt, Christopher R; Ng, Linda W C; Tsui, Wendy W Y; Leung, Suet Y; Yuen, Siu T; Becker, Natalia; Weichenhan, Dieter; Plass, Christoph; Schmezer, Peter; Chan, Tsun L; Popanda, Odilia

    2014-04-15

    Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.

  12. DNA Ligase IV Guides End-Processing Choice during Nonhomologous End Joining.

    PubMed

    Conlin, Michael P; Reid, Dylan A; Small, George W; Chang, Howard H; Watanabe, Go; Lieber, Michael R; Ramsden, Dale A; Rothenberg, Eli

    2017-09-19

    Nonhomologous end joining (NHEJ) must adapt to diverse end structures during repair of chromosome breaks. Here, we investigate the mechanistic basis for this flexibility. DNA ends are aligned in a paired-end complex (PEC) by Ku, XLF, XRCC4, and DNA ligase IV (LIG4); we show by single-molecule analysis how terminal mispairs lead to mobilization of ends within PECs and consequent sampling of more end-alignment configurations. This remodeling is essential for direct ligation of damaged and mispaired ends during cellular NHEJ, since remodeling and ligation of such ends both require a LIG4-specific structural motif, insert1. Insert1 is also required for PEC remodeling that enables nucleolytic processing when end structures block direct ligation. Accordingly, cells expressing LIG4 lacking insert1 are sensitive to ionizing radiation. Cellular NHEJ of diverse ends thus identifies the steps necessary for repair through LIG4-mediated sensing of differences in end structure and consequent dynamic remodeling of aligned ends. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  13. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

    PubMed Central

    Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

    2016-01-01

    The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity. PMID:26964677

  14. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface.

    PubMed

    Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

    2016-03-11

    The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference-NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

  15. Structure-Based Virtual Ligand Screening on the XRCC4/DNA Ligase IV Interface

    NASA Astrophysics Data System (ADS)

    Menchon, Grégory; Bombarde, Oriane; Trivedi, Mansi; Négrel, Aurélie; Inard, Cyril; Giudetti, Brigitte; Baltas, Michel; Milon, Alain; Modesti, Mauro; Czaplicki, Georges; Calsou, Patrick

    2016-03-01

    The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference - NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.

  16. DNA ligase IV and artemis act cooperatively to suppress homologous recombination in human cells: implications for DNA double-strand break repair.

    PubMed

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.

  17. DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

    PubMed Central

    Kurosawa, Aya; Saito, Shinta; So, Sairei; Hashimoto, Mitsumasa; Iwabuchi, Kuniyoshi; Watabe, Haruka; Adachi, Noritaka

    2013-01-01

    Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR. PMID:23967291

  18. A High-Throughput Scintillation Proximity-Based Assay for Human DNA Ligase IV

    PubMed Central

    Tseng, Hui-Min; Shum, David; Bhinder, Bhavneet; Escobar, Sindy; Veomett, Nicholas J.; Tomkinson, Alan E.; Gin, David Y.

    2012-01-01

    Abstract Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α-33P]-AMP from the complex Lig4-[α-33P]-AMP onto the 5′ end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z′ value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC50 values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors. PMID:22192310

  19. DNA ligase I, the replicative DNA ligase.

    PubMed

    Howes, Timothy R L; Tomkinson, Alan E

    2012-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication.

  20. DNA-ligase IV and DNA-protein kinase play a critical role in deficient caspases activation in apoptosis-resistant cancer cells by using doxorubicin.

    PubMed

    Friesen, Claudia; Uhl, Miriam; Pannicke, Ulrich; Schwarz, Klaus; Miltner, Erich; Debatin, Klaus-Michael

    2008-08-01

    Resistance toward cytotoxic drugs is one of the primary causes for therapeutic failure in cancer therapy. DNA repair mechanisms as well as deficient caspases activation play a critical role in apoptosis resistance of tumor cells toward anticancer drug treatment. Here, we discovered that deficient caspases activation in apoptosis-resistant cancer cells depends on DNA-ligase IV and DNA-protein kinase (DNA-PK), playing crucial roles in the nonhomologous end joining (NHEJ) pathway, which is the predominant pathway for DNA double-strand break repair (DNA-DSB-repair) in mammalian cells. DNA-PK(+/+) as well as DNA-ligase IV (+/+) cancer cells were apoptosis resistant and deficient in activation of caspase-3, caspase-9, and caspase-8 and in cleavage of poly(ADP-ribose) polymerase after doxorubicin treatment. Inhibition of NHEJ by knocking out DNA-PK or DNA-ligase IV restored caspases activation and apoptosis sensitivity after doxorubicin treatment. In addition, inhibition of caspases activation prevented doxorubicin-induced apoptosis but could not prevent doxorubicin-induced DNA damage, indicating that induction of DNA damage is independent of caspases activation. However, caspases activation depends on induction of DNA damage left unrepaired by NHEJ-DNA-DSB-repair. We conclude that DNA damage left unrepaired by DNA-ligase IV or DNA-PK might be the initiator for caspases activation by doxorubicin in cancer cells. Failure in caspases activation using doxorubicin depends on loss of DNA damage and is due to higher rates of NHEJ-DNA-DBS-repair.

  1. The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair.

    PubMed

    Pellarin, Ilenia; Arnoldo, Laura; Costantini, Silvia; Pegoraro, Silvia; Ros, Gloria; Penzo, Carlotta; Triolo, Gianluca; Demarchi, Francesca; Sgarra, Riccardo; Vindigni, Alessandro; Manfioletti, Guidalberto

    2016-01-01

    The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.

  2. The Architectural Chromatin Factor High Mobility Group A1 Enhances DNA Ligase IV Activity Influencing DNA Repair

    PubMed Central

    Costantini, Silvia; Pegoraro, Silvia; Ros, Gloria; Penzo, Carlotta; Triolo, Gianluca; Demarchi, Francesca; Sgarra, Riccardo; Vindigni, Alessandro; Manfioletti, Guidalberto

    2016-01-01

    The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens. PMID:27723831

  3. DNA ligases as therapeutic targets

    PubMed Central

    Tomkinson, Alan E.; Howes, Timothy R.L.; Wiest, Nathaniel E.

    2013-01-01

    During DNA replication, DNA joining events link Okazaki fragments on the lagging strand. In addition, they are required to repair DNA single- and double-strand breaks and to complete repair events initiated by the excision of mismatched and damaged bases. In human cells, there are three genes encoding DNA ligases. These enzymes are ATP-dependent and contain a conserved catalytic region. Biophysical studies have shown that the catalytic region contains three domains that, in the absence of DNA, are in an extended conformation. When the catalytic region engages a DNA nick, it adopts a compact, ring structure around the DNA nick with each of the three domains contacting the DNA. Protein-protein interactions involving the regions flanking the conserved catalytic regions of human DNA ligases play a major role in directing these enzymes to participate in specific DNA transactions. Among the human LIG genes, the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different N- and C-termini. One of these polypeptides is targeted to mitochondria where it plays an essential role in the maintenance of the mitochondrial genome. In the nucleus, DNA ligases I, III and IV have distinct but overlapping functions in DNA replication and repair. Small molecule inhibitors of human DNA ligases have been identified using structure-based approaches. As expected, these inhibitors are cytotoxic and also potentiate the cytotoxicity of DNA damaging agents. The results of preclinical studies with human cancer cell lines and mouse models of human cancer suggest that DNA ligase inhibitors may have utility as anti-cancer agents. PMID:24224145

  4. Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines.

    PubMed

    Nishida, Yayoi; Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Nozawa, Yoshinori; Minami, Yosuke; Ohnishi, Kazunori; Naoe, Tomoki; Murate, Takashi

    2014-01-01

    Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype.

  5. Structure of the catalytic region of DNA ligase IV in complex with an Artemis fragment sheds light on double-strand break repair.

    PubMed

    Ochi, Takashi; Gu, Xiaolong; Blundell, Tom L

    2013-04-02

    Nonhomologous end joining (NHEJ) is central to the repair of double-stranded DNA breaks throughout the cell cycle and plays roles in the development of the immune system. Although three-dimensional structures of most components of NHEJ have been defined, those of the catalytic region of DNA ligase IV (LigIV), a specialized DNA ligase known to work in NHEJ, and of Artemis have remained unresolved. Here, we report the crystal structure at 2.4 Å resolution of the catalytic region of LigIV (residues 1-609) in complex with an Artemis peptide. We describe interactions of the DNA-binding domain of LigIV with the continuous epitope of Artemis, which, together, form a three-helix bundle. A kink in the first helix of LigIV introduced by a conserved VPF motif gives rise to a hydrophobic pocket, which accommodates a conserved tryptophan from Artemis. We provide structural insights into features of LigIV among human DNA ligases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Prokaryotic DNA ligases unwind superhelical DNA.

    PubMed

    Ivanchenko, M; van Holde, K; Zlatanova, J

    1996-09-13

    We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E. coli) to superhelical or nicked circular DNA. Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values. This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E. coli DNA ligase in the presence of increasing amounts of T4 ligase. Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity. Performing a similar experiment with reverse partners, using E. coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E. coli enzyme also unwound the DNA. Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

  7. Novel compound heterozygous DNA ligase IV mutations in an adolescent with a slowly-progressing radiosensitive-severe combined immunodeficiency.

    PubMed

    Tamura, Shinobu; Higuchi, Kohei; Tamaki, Masaharu; Inoue, Chizuko; Awazawa, Ryoko; Mitsuki, Noriko; Nakazawa, Yuka; Mishima, Hiroyuki; Takahashi, Kenzo; Kondo, Osamu; Imai, Kohsuke; Morio, Tomohiro; Ohara, Osamu; Ogi, Tomoo; Furukawa, Fukumi; Inoue, Masami; Yoshiura, Koh-ichiro; Kanazawa, Nobuo

    2015-10-01

    We herein describe a case of a 17-year-old boy with intractable common warts, short stature, microcephaly and slowly-progressing pancytopenia. Simultaneous quantification of T-cell receptor recombination excision circles (TREC) and immunoglobulin κ-deleting recombination excision circles (KREC) suggested very poor generation of both T-cells and B-cells. By whole exome sequencing, novel compound heterozygous mutations were identified in the patient's DNA ligase IV (LIG4) gene. The diagnosis of LIG4 syndrome was confirmed by delayed DNA double-strand break repair kinetics in γ-irradiated fibroblasts from the patient and their restoration by an introduction of wild-type LIG4. Although the patient received allogeneic hematopoietic stem cell transplantation from his haploidentical mother, he unfortunately expired due to an insufficiently reconstructed immune system. An earlier definitive diagnosis using TREC/KREC quantification and whole exome sequencing would thereby allow earlier intervention, which would be essential for improving long-term survival in similar cases with slowly-progressing LIG4 syndrome masked in adolescents.

  8. An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

    SciTech Connect

    Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; Chokshi, Chirayu; Tsai, Miaw-Sheue; Matsumoto, Yoshihiro; Kuzdovich, Monica; Remesh, Soumya G.; Fang, Shujuan; Tomkinson, Alan E.; Lees-Miller, Susan P.; Tainer, John A.

    2016-11-14

    DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.

  9. An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

    DOE PAGES

    Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; ...

    2016-11-14

    DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

  10. An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex*

    PubMed Central

    Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; Chokshi, Chirayu; Tsai, Miaw-Sheue; Matsumoto, Yoshihiro; Kuzdovich, Monica; Remesh, Soumya G.; Fang, Shujuan; Tomkinson, Alan E.; Tainer, John A.

    2016-01-01

    DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes. PMID:27875301

  11. An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex.

    PubMed

    Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K; Chokshi, Chirayu; Tsai, Miaw-Sheue; Matsumoto, Yoshihiro; Kuzdovich, Monica; Remesh, Soumya G; Fang, Shujuan; Tomkinson, Alan E; Lees-Miller, Susan P; Tainer, John A

    2016-12-30

    DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). Yet, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcs (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.

    PubMed

    Park, Jihye; Welner, Robert S; Chan, Mei-Yee; Troppito, Logan; Staber, Philipp B; Tenen, Daniel G; Yan, Catherine T

    2016-01-01

    Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients. Copyright © 2015 by The American Association of Immunologists, Inc.

  13. Gene targeting by RNAi-mediated knockdown of potent DNA ligase IV homologue in the cellulase-producing fungus Talaromyces cellulolyticus.

    PubMed

    Hayata, Koutarou; Asada, Seiya; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Sawayama, Shigeki

    2014-11-01

    The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

  14. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV.

    PubMed

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin; Ding, Nan; Qi, Yongmei; Zhang, Yingmei; Wang, Jufang; Huang, Dejun

    2015-09-01

    Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1-8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. THE HUMAN SET AND TRANSPOSASE DOMAIN PROTEIN METNASE INTERACTS WITH DNA LIGASE IV AND ENHANCES THE EFFICIENCY AND ACCURACY OF NON-HOMOLOGOUS END JOINING

    PubMed Central

    Hromas, Robert; Wray, Justin; Lee, Suk-Hee; Martinez, Leah; Farrington, Jacqueline; Corwin, Lori Kwan; Ramsey, Heather; Nickoloff, Jac A.; Williamson, Elizabeth A.

    2008-01-01

    Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and Transposase domain protein termed Metnase that promotes DNA double strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and Transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of γ-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs. PMID:18773976

  16. The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining.

    PubMed

    Hromas, Robert; Wray, Justin; Lee, Suk-Hee; Martinez, Leah; Farrington, Jacqueline; Corwin, Lori Kwan; Ramsey, Heather; Nickoloff, Jac A; Williamson, Elizabeth A

    2008-12-01

    Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of gamma-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.

  17. Expression of DNA ligase IV is linked to poor prognosis and characterizes a subset of prostate cancers harboring TMPRSS2:ERG fusion and PTEN deletion.

    PubMed

    Grupp, Katharina; Roettger, Laura; Kluth, Martina; Hube-Magg, Claudia; Simon, Ronald; Lebok, Patrick; Minner, Sarah; Tsourlakis, Maria Christina; Koop, Christina; Graefen, Markus; Adam, Meike; Haese, Alexander; Wittmer, Corinna; Sauter, Guido; Wilczak, Waldemar; Huland, Hartwig; Schlomm, Thorsten; Steurer, Stefan; Krech, Till

    2015-09-01

    DNA ligases are essential for the maintenance of genome integrity as they are indispensable for DNA replication, recombination and repair. The present study was undertaken to gain insights into the prevalence and clinical significance of ligase IV (LIG4) expression in prostate cancer. A total of 11,152 prostate cancer specimens were analyzed by immunohistochemistry for LIG4 expression. Results were compared to follow-up data, ERG status and deletions at PTEN, 3p13, 5q21 and 6q15. LIG4 expression was predominantly localized in the nucleus of the cells with increased intensities in malignant as compared to benign prostate epithelium. In prostate cancer, LIG4 expression was found in 91% of interpretable tumors, including 12% cancers with weak, 23% with moderate and 56% with strong LIG4 positivity. Strong LIG4 expression was tightly linked to advanced Gleason score (P<0.0001) and positive nodal involvement (P=0.03). There was a remarkable accumulation of strong LIG4 expression in tumors harboring TMPRSS2:ERG fusion and PTEN deletions (P<0.0001 each). High LIG4 expression was also tightly related to early biochemical recurrence when all tumors (P<0.0001) or the subsets of ERG-negative (P=0.0004) or ERG-positive prostate cancers (P=0.006) were analyzed. Multivariate analysis including parameters that are available before surgery demonstrated independent association with biochemical recurrence for advanced Gleason grade on biopsy, high preoperative PSA level, high clinical stage (P<0.0001 each) and for LIG4 immunostaining (P=0.03). Our study identifies LIG4 as a predictor of an increased risk for early PSA recurrence in prostate cancer. Moreover, the strong association with TMPRSS2:ERG fusion and PTEN deletions suggest important interactions between these pathways in prostate cancers.

  18. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    PubMed

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  19. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene.

    PubMed

    Tomkinson, Alan E; Sallmyr, Annahita

    2013-12-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. © 2013 Elsevier B.V. All rights reserved.

  20. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    PubMed Central

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase IIIα is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. Unlike its other nuclear functions, the role of DNA ligase IIIα in alternative NHEJ is independent of its nuclear partner protein, X-ray repair cross-complementing protein 1 (XRCC1). DNA ligase IIIα is frequently overexpressed in cancer cells, acting as a biomarker for increased dependence upon alternative NHEJ for DSB repair and it is a promising novel therapeutic target. PMID:24013086

  1. Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV.

    PubMed

    Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa

    2015-06-12

    XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4(K271R) mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4(K210R) mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3'-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4(K271R) was defective in the nuclear localization of itself and LIG4, whereas XRCC4(K210R) was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4(WT). The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4(K271R) than in M10-XRCC4(WT), whereas it was only marginally increased in M10-XRCC4(K210R) as compared to M10-XRCC4(WT). The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4.

  2. DNA looping by a ligase under nanoconfinement

    NASA Astrophysics Data System (ADS)

    Heidarpour-Roushan, Maedeh; Riehn, Robert

    2013-03-01

    DNA looping is essential for the function and maintenance of genetic information. We have investigated the kinetic evolution of DNA loops (48500 bp) induced by T4 ligase inside a nanofabricated channel system with a channel cross-section of 100x100 nm2, and a few hundred microns channel length. We found that addition of the ligase profoundly alters the behavior of DNA. In particular, ligase acts to stabilize hairpin geometries in which the extended forward and backward arms of the hairpin scan past each other. From the linear density of DNA inside the channel, we deduce that the effective excluded volume vanishes upon addition of T4 ligase and ATP. We conclude that the two strands are effectively stapled together through a large number of weak bonds involving T4 ligase.

  3. Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

    SciTech Connect

    Fukuchi, Mikoto; Wanotayan, Rujira; Liu, Sicheng; Imamichi, Shoji; Sharma, Mukesh Kumar; Matsumoto, Yoshihisa

    2015-06-12

    XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4{sup K271R} mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4{sup K210R} mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4{sup K271R} was defective in the nuclear localization of itself and LIG4, whereas XRCC4{sup K210R} was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4{sup K271R}, but not M10-XRCC4{sup K210R}, showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4{sup WT}. The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4{sup K271R} than in M10-XRCC4{sup WT}, whereas it was only marginally increased in M10-XRCC4{sup K210R} as compared to M10-XRCC4{sup WT}. The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4. - Highlights: • XRCC4{sup K271R} is defective in the nuclear localization of itself and LIG4. • XRCC4{sup K210R} is competent for the nuclear localization of itself and LIG4. • XRCC4{sup K271R} is deficient in DSB repair function. • XRCC4{sup K210R} is mostly normal in DSB repair function.

  4. Disconnecting XRCC1 and DNA ligase III.

    PubMed

    Katyal, Sachin; McKinnon, Peter J

    2011-07-15

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease.

  5. Disconnecting XRCC1 and DNA ligase III

    PubMed Central

    Katyal, Sachin

    2011-01-01

    DNA strand break repair is essential for the prevention of multiple human diseases, particularly those which feature neuropathology. To further understand the pathogenesis of these syndromes, we recently developed animal models in which the DNA single-strand break repair (SSBR) components, XRCC1 and DNA Ligase III (LIG3), were inactivated in the developing nervous system. Although biochemical evidence suggests that inactivation of XRCC1 and LIG3 should share common biological defects, we found profound phenotypic differences between these two models, implying distinct biological roles for XRCC1 and LIG3 during DNA repair. Rather than a key role in nuclear DNA repair, we found LIG3 function was central to mitochondrial DNA maintenance. Instead, our data indicate that DNA Ligase 1 is the main DNA ligase for XRCC1-mediated DNA repair. These studies refine our understanding of DNA SSBR and the etiology of neurological disease. PMID:21636980

  6. Poly (ADP-Ribose) Polymerase is Involved in the Repair of DNA Damage Due to Sulfur Mustard by a Mechanism Other Than DNA Ligase I Activation

    DTIC Science & Technology

    2004-11-16

    agents including sulfur mustard (SM). We observed concurrent activation of PARP and DNA ligase in SM-exposed human epidermal keratinocytes (HEK...Previous reports from other laboratories suggested that DNA ligase activation could be due to its modification by PARP. In humans, there are three distinct...DNA ligases, I, II and IV of which DNA ligase I participates in DNA replication and repair. By metabolically labeling HEK using 3H-adenosine

  7. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding

    PubMed Central

    Chiruvella, Kishore K.; Renard, Brian M.; Birkeland, Shanda R.; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E.

    2014-01-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure-function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4-Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association. PMID:25457772

  8. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding.

    PubMed

    Chiruvella, Kishore K; Renard, Brian M; Birkeland, Shanda R; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E

    2014-12-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure–function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4–Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association.

  9. Engineered DNA ligases with improved activities in vitro.

    PubMed

    Wilson, Robert H; Morton, Susan K; Deiderick, Heather; Gerth, Monica L; Paul, Hayden A; Gerber, Ilana; Patel, Ankita; Ellington, Andrew D; Hunicke-Smith, Scott P; Patrick, Wayne M

    2013-07-01

    The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.

  10. DNA and RNA ligases: structural variations and shared mechanisms.

    PubMed

    Pascal, John M

    2008-02-01

    DNA and RNA ligases join 3' OH and 5' PO4 ends in polynucleotide substrates using a three-step reaction mechanism that involves covalent modification of both the ligase enzyme and the polynucleotide substrate with AMP. In the past three years, several polynucleotide ligases have been crystallized in complex with nucleic acid, providing the introductory views of ligase enzymes engaging their substrates. Crystal structures for two ATP-dependent DNA ligases, an NAD+-dependent DNA ligase, and an ATP-dependent RNA ligase demonstrate how ligases utilize the AMP group and their multi-domain architectures to manipulate nucleic acid structure and catalyze the end-joining reaction. Together with unliganded crystal structures of DNA and RNA ligases, a more comprehensive and dynamic understanding of the multi-step ligation reaction mechanism has emerged.

  11. Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

    PubMed Central

    Aoufouchi, S; Hardy, S; Prigent, C; Philippe, M; Thiebaud, P

    1991-01-01

    We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules. Images PMID:1886765

  12. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.

    PubMed

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-02-02

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4(-/-) cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.

  13. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

    PubMed

    Lohman, Gregory J S; Zhang, Yinhua; Zhelkovsky, Alexander M; Cantor, Eric J; Evans, Thomas C

    2014-02-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

  14. Expression and biochemical characterization of Plasmodium falciparum DNA ligase I.

    PubMed

    Buguliskis, Jeffrey S; Casta, Louis J; Butz, Charles E; Matsumoto, Yoshihiro; Taraschi, Theodore F

    2007-10-01

    We report that Plasmodium falciparum (Pf) encodes a 912 amino acid ATP-dependent DNA ligase. Protein sequence analysis of Pf DNA ligase I indicates a strong sequence similarity, particularly in the C-terminal region, to DNA ligase I homologues. The activity of recombinant Pf DNA ligase I (PfLigI) was investigated using protein expressed in HEK293 cells. The PfLigI gene product is approximately 94kDa and catalyzes phosphodiester bond formation on a singly nicked DNA substrate. The enzyme is most active at alkaline pH (8.5) and with Mg(2+) or Mn(2+) and ATP as cofactors. Kinetic studies of PfLigI revealed that the enzyme has similar substrate affinity (K(m) 2.6nM) as compared to human DNA ligase I and k(cat) (2.3x10(-3)s(-1)) and k(cat)/K(m) (8.8x10(5)M(-1)s(-1)) which are similar to other ATP-dependent DNA ligases. PfLigI was able to join RNA-DNA substrates only when the RNA sequence was upstream of the nick, confirming that it is DNA ligase I and has no associated DNA ligase III like activity.

  15. DNA Ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair

    PubMed Central

    Gao, Yankun; Katyal, Sachin; Lee, Youngsoo; Zhao, Jingfeng; Rehg, Jerold E.; Russell, Helen R.; McKinnon, Peter J.

    2011-01-01

    DNA replication and repair in mammalian cells involves three distinct DNA ligases; ligase I (Lig1), ligase III (Lig3) and ligase IV (Lig4)1. Lig3 is considered a key ligase during base excision repair because its stability depends upon its nuclear binding partner Xrcc1, a critical factor for this DNA repair pathway2,3. Lig3 is also present in the mitochondria where its role in mitochondrial DNA (mtDNA) maintenance is independent of Xrcc14. However, the biological role of Lig3 is unclear as inactivation of murine Lig3 results in early embryonic lethality5. Here we report that Lig3 is essential for mtDNA integrity but dispensable for nuclear DNA repair. Inactivation of Lig3 in the mouse nervous system resulted in mtDNA loss leading to profound mitochondrial dysfunction, disruption of cellular homeostasis and incapacitating ataxia. Similarly, inactivation of Lig3 in cardiac muscle resulted in mitochondrial dysfunction and defective heart pump function leading to heart failure. However, Lig3 inactivation did not result in nuclear DNA repair deficiency, indicating essential DNA repair functions of Xrcc1 can occur in the absence of Lig3. Instead, we found that Lig1 was critical for DNA repair, but in a cooperative manner with Lig3. Additionally, Lig3 deficiency did not recapitulate the hallmark features of neural Xrcc1 inactivation such as DNA damage-induced cerebellar interneuron loss6, further underscoring functional separation of these DNA repair factors. Therefore, our data reveal that the critical biological role of Lig3 is to maintain mtDNA integrity and not Xrcc1-dependent DNA repair. PMID:21390131

  16. Specific Inhibition of the Eubacterial DNA Ligase by Arylamino Compounds

    PubMed Central

    Ciarrocchi, Giovanni; MacPhee, Donald G.; Deady, Les W.; Tilley, Leann

    1999-01-01

    All known DNA ligases catalyze the formation of a phosphodiester linkage between adjacent termini in double-stranded DNA via very similar mechanisms. The ligase family can, however, be divided into two classes: eubacterial ligases, which require NAD+ as a cofactor, and other ligases, from viruses, archaea, and eukaryotes, which use ATP. Drugs that discriminate between DNA ligases from different sources may have antieubacterial activity. We now report that a group of arylamino compounds, including some commonly used antimalarial and anti-inflammatory drugs and a novel series of bisquinoline compounds, are specific inhibitors of eubacterial DNA ligases. Members of this group of inhibitors have different heterocyclic ring systems with a common amino side chain in which the two nitrogens are separated by four carbon atoms. The potency, but not the specificity of action, is influenced by the DNA-binding characteristics of the inhibitor, and the inhibition is noncompetitive with respect to NAD+. The arylamino compounds appear to target eubacterial DNA ligase in vivo, since a Salmonella Lig− strain that has been rescued with the ATP-dependent T4 DNA ligase is less sensitive than the parental Salmonella strain. PMID:10543760

  17. Human DNA ligases: a comprehensive new look for cancer therapy.

    PubMed

    Singh, Deependra Kumar; Krishna, Shagun; Chandra, Sharat; Shameem, Mohammad; Deshmukh, Amit Laxmikant; Banerjee, Dibyendu

    2014-05-01

    Living organisms belonging to all three domains of life, viz., eubacteria, archaeabacteria, and eukaryotes encode one or more DNA ligases. DNA ligases are indispensable in various DNA repair and replication processes and a deficiency or an inhibition of their activity can lead to accumulation of DNA damage and strand breaks. DNA damage, specially strand breaks at unsustainable levels can lead to replication block and/or cell death. DNA ligases as potential anticancer targets have been realized only recently. There is enough rationale to suggest that ligases have a tremendous potential for novel therapeutics including anticancer and antibacterial therapy, specially when the world is facing acute problems of drug resistance and chemotherapy failure, with an immediate need for new therapeutic targets. Here, we review the current state of the art in the development of human ligase inhibitors, their structures, molecular mechanisms, physiological effects, and their potential in future cancer therapy. Citing examples, we focus on strategies for improving the activity and specificity of existing and novel inhibitors by using structure-based rational approaches. In the end, we describe potential new sites on the ligase I protein that can be targeted for the development of novel inhibitors. This is the first comprehensive review to compile all known human ligase inhibitors and to provide a rationale for the further development of ligase inhibitors for cancer therapy. © 2013 Wiley Periodicals, Inc.

  18. Cellular DNA ligase I is recruited to cytoplasmic vaccinia virus factories and masks the role of the vaccinia ligase in viral DNA replication.

    PubMed

    Paran, Nir; De Silva, Frank S; Senkevich, Tatiana G; Moss, Bernard

    2009-12-17

    Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. RNA silencing of human DNA ligase I expression and a small molecule inhibitor of human DNA ligase I [corrected] severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding its own ligase might allow VACV to "jump-start" DNA synthesis.

  19. DNA ligase I is not essential for mammalian cell viability.

    PubMed

    Han, Li; Masani, Shahnaz; Hsieh, Chih-lin; Yu, Kefei

    2014-04-24

    Of the three DNA ligases present in all vertebrates, DNA ligase I (Lig1) has been considered essential for ligating Okazaki fragments during DNA replication and thereby essential for cell viability. Here, we report the striking finding that a Lig1-null murine B cell line is viable. Surprisingly, the Lig1-null cells exhibit normal proliferation and normal immunoglobulin heavy chain class switch recombination and are not hypersensitive to a wide variety of DNA damaging agents. These findings demonstrate that Lig1 is not absolutely required for cellular DNA replication and repair and that either Lig3 or Lig4 can substitute for the role of Lig1 in joining Okazaki fragments. The establishment of a Lig1-null cell line will greatly facilitate the characterization of DNA ligase function in mammalian cells, but the finding alone profoundly reprioritizes the role of ligase I in DNA replication, repair, and recombination.

  20. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    PubMed

    Arakawa, Hiroshi; Iliakis, George

    2015-06-23

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

  1. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase.

    PubMed

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-08-21

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246-401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95-G96 and containing 5'-phosphomonoester and 3'-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively.

  2. Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase.

    PubMed

    Wang, You; Xie, Juan-Juan; Han, Zhong; Liu, Jian-Hua; Liu, Xi-Peng

    2013-02-01

    We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.

  3. Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states.

    PubMed

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A; Tomkinson, Alan E; Ellenberger, Tom

    2010-07-27

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  4. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    SciTech Connect

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  5. Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining

    PubMed Central

    Lu, Guangqing; Duan, Jinzhi; Shu, Sheng; Wang, Xuxiang; Gao, Linlin; Guo, Jing; Zhang, Yu

    2016-01-01

    In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4−/− cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ. PMID:26787905

  6. DNA ligase and the pyridine nucleotide cycle in Salmonella typhimurium.

    PubMed Central

    Park, U E; Olivera, B M; Hughes, K T; Roth, J R; Hillyard, D R

    1989-01-01

    Bacterial DNA ligases use NAD as an energy source. In this study we addressed two questions about these enzymes. First, what is the physiological consequence of completely removing the NAD-dependent enzyme and replacing it with an ATP-dependent DNA ligase? We constructed Salmonella typhimurium strains in which the endogenous NAD-dependent DNA ligase activity was inactivated by an insertion mutation and the ATP-dependent enzyme from bacteriophage T4 was provided by a cloned phage gene. Such strains were physiologically indistinguishable from the wild type, even under conditions of UV irradiation or treatment with alkylating agents. These results suggest that specific functional interactions between DNA ligase and other replication and repair enzymes may be unimportant under the conditions tested. Second, the importance of DNA ligation as the initiating event of the bacterial pyridine nucleotide cycle was critically assessed in these mutant strains. Surprisingly, our results indicate that DNA ligation makes a minimal contribution to the pyridine nucleotide cycle; the Salmonella strains with only an ATP-dependent ligase had the same NAD turnover rates as the wild-type strain with an NAD-dependent ligase. However, we found that NAD turnover was significantly decreased under anaerobic conditions. We suggest that most intracellular pyridine nucleotide breakdown occurs in a process that protects the cell against oxygen damage but involves a biochemical mechanism other than DNA ligation. Images PMID:2649488

  7. T4 DNA ligase is more than an effective trap of cyclized dsDNA.

    PubMed

    Yuan, Chongli; Lou, Xiong Wen; Rhoades, Elizabeth; Chen, Huimin; Archer, Lynden A

    2007-01-01

    T4 DNA ligase is used in standard cyclization assays to trap double-stranded DNA (dsDNA) in low-probability, cyclic or highly bent conformations. The cyclization probability, deduced from the relative yield of cyclized product, can be used in conjunction with statistical mechanical models to extract the bending stiffness of dsDNA. By inserting the base analog 2-aminopurine (2-AP) at designated positions in 89 bp and 94 bp dsDNA fragments, we find that T4 DNA ligase can have a previously unknown effect. Specifically, we observe that addition of T4 ligase to dsDNA in proportions comparable to what is used in the cyclization assay leads to a significant increase in fluorescence from 2-AP. This effect is believed to originate from stabilization of local base-pair opening by formation of transient DNA-ligase complexes. Non-specific binding of T4 ligase to dsDNA is also confirmed using fluorescence correlation spectroscopy (FCS) experiments, which reveal a systematic reduction of dsDNA diffusivity in the presence of ligase. ATP competes with regular DNA for non-covalent binding to the T4 ligase and is found to significantly reduce DNA-ligase complexation. For short dsDNA fragments, however, the population of DNA-ligase complexes at typical ATP concentrations used in DNA cyclization studies is determined to be large enough to dominate the cyclization reaction.

  8. Biochemical characterization of the DNA ligase I from Entamoeba histolytica.

    PubMed

    Cardona-Felix, Cesar S; Pastor-Palacios, Guillermo; Cardenas, Helios; Azuara-Liceaga, Elisa; Brieba, Luis G

    2010-11-01

    DNA ligases play an essential role in DNA replication and repair. Herein, we report the cloning and biochemical characterization of DNA ligase I from the protozoan parasite Entamoeba histolytica (EhDNAligI). EhDNAligI is an ATP-dependent DNA ligase of 685 amino acids with 35% identity to human DNA ligase I. This report shows that heterologous expressed EhDNAligI is able to perform the three conserved steps of a DNA ligation reaction: adenylation, binding to a 5'-phosphorylated nicked DNA substrate and sealing of the nick. EhDNAligI is strongly inhibited by NaCl and displays optimal activity at pH 7.5. EhDNAligI uses Mn2+ or Mg2+ as metal cofactors and ATP as nucleotide cofactor. EhDNAligI has a nicked DNA binding constant of 6.6microM and follows Michaelis-Menten steady-state kinetics with a K(m) ATP of 64nM and a k(cat) of 2.4min(-1). Accordingly to its properties as a family I DNA ligase, EhDNAligI is able to ligate a RNA strand upstream of a nucleic acid nick, but not in the downstream or the template position. We propose that EhDNAligI is involved in sealing DNA nicks during lagging strand synthesis and may have a role in base excision repair in E. histolytica.

  9. Polynucleotide 3'-terminal phosphate modifications by RNA and DNA ligases.

    PubMed

    Zhelkovsky, Alexander M; McReynolds, Larry A

    2014-11-28

    RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3'-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3'p substrate to generate an RNA 2',3'-cyclic phosphate or convert DNA3'p to ssDNA(3')pp(5')A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3'p. These modifications of RNA and DNA 3'-phosphates are similar to the activities of RtcA, an RNA 3'-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3'p or DNA 3'p to generate the adenylated intermediate. For RNA (3')pp(5')A, the third step involves attack of the adjacent 2' hydroxyl to generate the RNA 2',3'-cyclic phosphate. These steps are analogous to those in classical 5' phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3'p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3'-phosphorylated nicks in double-stranded DNA to produce a 3'-adenylated product. These 3'-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5'Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3'-terminal phosphates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase

    PubMed Central

    Lohman, Gregory J. S.; Zhang, Yinhua; Zhelkovsky, Alexander M.; Cantor, Eric J.; Evans, Thomas C.

    2014-01-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA. PMID:24203707

  11. Rapid Assembly of DNA via Ligase Cycling Reaction (LCR).

    PubMed

    Chandran, Sunil

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the ligase chain reaction (LCR). The LCR method utilizes non-overlapping DNA parts that are ligated together with the guidance of bridging oligos. Using this method, we have successfully assembled up to 20 DNA parts in a single reaction or DNA constructs up to 26 kb in size.

  12. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase

    PubMed Central

    Nohales, María-Ángeles; Flores, Ricardo; Daròs, José-Antonio

    2012-01-01

    Viroids are a unique class of noncoding RNAs: composed of only a circular, single-stranded molecule of 246–401 nt, they manage to replicate, move, circumvent host defenses, and frequently induce disease in higher plants. Viroids replicate through an RNA-to-RNA rolling-circle mechanism consisting of transcription of oligomeric viroid RNA intermediates, cleavage to unit-length strands, and circularization. Though the host RNA polymerase II (redirected to accept RNA templates) mediates RNA synthesis and a type-III RNase presumably cleavage of Potato spindle tuber viroid (PSTVd) and closely related members of the family Pospiviroidae, the host enzyme catalyzing the final circularization step, has remained elusive. In this study we propose that PSTVd subverts host DNA ligase 1, converting it to an RNA ligase, for the final step. To support this hypothesis, we show that the tomato (Solanum lycopersicum L.) DNA ligase 1 specifically and efficiently catalyzes circularization of the genuine PSTVd monomeric linear replication intermediate opened at position G95–G96 and containing 5′-phosphomonoester and 3′-hydroxyl terminal groups. Moreover, we also show a decreased PSTVd accumulation and a reduced ratio of monomeric circular to total monomeric PSTVd forms in Nicotiana benthamiana Domin plants in which the endogenous DNA ligase 1 was silenced. Thus, in a remarkable example of parasitic strategy, viroids reprogram for their replication the template and substrate specificity of a DNA-dependent RNA polymerase and a DNA ligase to act as RNA-dependent RNA polymerase and RNA ligase, respectively. PMID:22869737

  13. Mammalian DNA ligase III: Molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination

    SciTech Connect

    Chen, Jingwen; Danehower, S.; Besterman, J.M.; Husain, I.

    1995-10-01

    Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating supermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replications. In contrast, elevated levels of DNA ligase III mRNA were observed in primary supermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells. 62 refs., 7 figs.

  14. DNA ligases ensure fidelity by interrogating minor groove contacts.

    PubMed

    Liu, Pingfang; Burdzy, Artur; Sowers, Lawrence C

    2004-01-01

    DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of

  15. Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I

    PubMed Central

    Srivastava, Sandeep Kumar; Dube, Divya; Tewari, Neetu; Dwivedi, Namrata; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2005-01-01

    DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD+-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD+ binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, Nn-bis-(5-deoxy-α-d-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD+ for binding and inhibit enzyme activity with IC50 values in the µM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD+ with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATP-dependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents. PMID:16361267

  16. Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I.

    PubMed

    Srivastava, Sandeep Kumar; Dube, Divya; Tewari, Neetu; Dwivedi, Namrata; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2005-01-01

    DNA ligases are important enzymes which catalyze the joining of nicks between adjacent bases of double-stranded DNA. NAD+-dependent DNA ligases (LigA) are essential in bacteria and are absent in humans. They have therefore been identified as novel, validated and attractive drug targets. Using virtual screening against an in-house database of compounds and our recently determined crystal structure of the NAD+ binding domain of the Mycobacterium tuberculosis LigA, we have identified N1, N(n)-bis-(5-deoxy-alpha-D-xylofuranosylated) diamines as a novel class of inhibitors for this enzyme. Assays involving M.tuberculosis LigA, T4 ligase and human DNA ligase I show that these compounds specifically inhibit LigA from M.tuberculosis. In vitro kinetic and inhibition assays demonstrate that the compounds compete with NAD+ for binding and inhibit enzyme activity with IC50 values in the microM range. Docking studies rationalize the observed specificities and show that among several glycofuranosylated diamines, bis xylofuranosylated diamines with aminoalkyl and 1, 3-phenylene carbamoyl spacers mimic the binding modes of NAD+ with the enzyme. Assays involving LigA-deficient bacterial strains show that in vivo inhibition of ligase by the compounds causes the observed antibacterial activities. They also demonstrate that the compounds exhibit in vivo specificity for LigA over ATP-dependent ligase. This class of inhibitors holds out the promise of rational development of new anti-tubercular agents.

  17. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

    PubMed

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

    2017-01-23

    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3' biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

  18. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    PubMed Central

    Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna

    2017-01-01

    Abstract DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude. PMID:28119419

  19. Heterogeneity of mammalian DNA ligase detected on activity and DNA sequencing gels.

    PubMed Central

    Mezzina, M; Sarasin, A; Politi, N; Bertazzoni, U

    1984-01-01

    A new method to detect DNA ligase activity in situ after NaDodSO4 polyacrylamide gel electrophoresis has been developed. After renaturation of active polypeptides the ligase reaction occurs in situ by incubating the intact gel in the presence of Mg++ and ATP. Further treatment with alkaline phosphatase removes the unligated 5'-32P-end of oligo (dT) used as a substrate and active polypeptides having ligase activity are identified by autoradiography. Analysis on DNA sequencing gels of the oligo (dT) reaction products present in the activity bands ensures that the radioactive material detected in activity gels or in standard in vitro ligase assays corresponds unambiguously to a ligase activity. Using these methods, we have analysed the purified phage T4 DNA ligase, and the activities present in crude extracts and in purified fractions from monkey kidney (CV1-P) cells. The purified T4 enzyme yields one or two active peptides with Mr values of 60,000 and 70,000. Crude extracts from CV1-P cells contain several polypeptides having DNA ligase activity. Partial purification of these extracts shows that DNA ligase I isolated from hydroxylapatite column is enriched in polypeptides with Mr 200,000, 150,000 and 120,000, while DNA ligase II is enriched in those with Mr 60,000 and 70,000. Images PMID:6377238

  20. One-step assay for the quantification of T4 DNA ligase.

    PubMed

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  1. Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells.

    PubMed

    Ortiz, T; Edreira, A; Piñero, J

    2002-06-01

    We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.

  2. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).

  3. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

    PubMed

    Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

    2011-04-01

    During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms

  4. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    PubMed

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  5. Splint ligation of RNA with T4 DNA ligase

    PubMed Central

    Kershaw, Christopher J.; O’Keefe, Raymond T.

    2014-01-01

    Splint ligation of RNA, whereby specific RNA molecules are ligated together, can be carried out using T4 DNA ligase and a bridging DNA oligonucleotide complementary to the RNAs. This method takes advantage of the property of T4 DNA ligase to join RNA molecules when they are in an RNA:DNA hybrid. Splint ligation is a useful tool for the introduction of modified nucleotides into RNA molecules, insertion of a radiolabel into a specific position within an RNA and for the assembly of smaller synthetic RNAs into longer RNA molecules. Such modifications enable a wide range of experiments to be carried out with the modified RNA including structural studies, co-immunoprecipitations, and the ability to map sites of RNA:RNA and RNA:protein interactions. PMID:23065567

  6. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    PubMed Central

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  7. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

    PubMed

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L; Tomkinson, Alan E; Tainer, John A; Ellenberger, Tom

    2015-08-18

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

  8. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    PubMed

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  9. DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.

    PubMed

    Vago, Riccardo; Leva, Valentina; Biamonti, Giuseppe; Montecucco, Alessandra

    2009-08-15

    DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.

  10. DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation.

    PubMed

    Simsek, Deniz; Brunet, Erika; Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J; Gregory, Philip D; Holmes, Michael C; Jasin, Maria

    2011-06-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1.

  11. DNA Ligase III Promotes Alternative Nonhomologous End-Joining during Chromosomal Translocation Formation

    PubMed Central

    Wong, Sunnie Yan-Wai; Katyal, Sachin; Gao, Yankun; McKinnon, Peter J.; Lou, Jacqueline; Zhang, Lei; Li, James; Rebar, Edward J.; Gregory, Philip D.; Holmes, Michael C.; Jasin, Maria

    2011-01-01

    Nonhomologous end-joining (NHEJ) is the primary DNA repair pathway thought to underlie chromosomal translocations and other genomic rearrangements in somatic cells. The canonical NHEJ pathway, including DNA ligase IV (Lig4), suppresses genomic instability and chromosomal translocations, leading to the notion that a poorly defined, alternative NHEJ (alt-NHEJ) pathway generates these rearrangements. Here, we investigate the DNA ligase requirement of chromosomal translocation formation in mouse cells. Mammals have two other DNA ligases, Lig1 and Lig3, in addition to Lig4. As deletion of Lig3 results in cellular lethality due to its requirement in mitochondria, we used recently developed cell lines deficient in nuclear Lig3 but rescued for mitochondrial DNA ligase activity. Further, zinc finger endonucleases were used to generate DNA breaks at endogenous loci to induce translocations. Unlike with Lig4 deficiency, which causes an increase in translocation frequency, translocations are reduced in frequency in the absence of Lig3. Residual translocations in Lig3-deficient cells do not show a bias toward use of pre-existing microhomology at the breakpoint junctions, unlike either wild-type or Lig4-deficient cells, consistent with the notion that alt-NHEJ is impaired with Lig3 loss. By contrast, Lig1 depletion in otherwise wild-type cells does not reduce translocations or affect microhomology use. However, translocations are further reduced in Lig3-deficient cells upon Lig1 knockdown, suggesting the existence of two alt-NHEJ pathways, one that is biased toward microhomology use and requires Lig3 and a back-up pathway which does not depend on microhomology and utilizes Lig1. PMID:21655080

  12. Rapid Time Scale Analysis of T4 DNA Ligase-DNA Binding.

    PubMed

    Bauer, Robert J; Jurkiw, Thomas J; Evans, Thomas C; Lohman, Gregory J S

    2017-02-28

    DNA ligases, essential to both in vivo genome integrity and in vitro molecular biology, catalyze phosphodiester bond formation between adjacent 3'-OH and 5'-phosphorylated termini in dsDNA. This reaction requires enzyme self-adenylylation, using ATP or NAD(+) as a cofactor, and AMP release concomitant with phosphodiester bond formation. In this study, we present the first fast time scale binding kinetics of T4 DNA ligase to both nicked substrate DNA (nDNA) and product-equivalent non-nicked dsDNA, as well as binding and release kinetics of AMP. The described assays utilized a fluorescein-dT labeled DNA substrate as a reporter for ligase·DNA interactions via stopped-flow fluorescence spectroscopy. The analysis revealed that binding to nDNA by the active adenylylated ligase occurs in two steps, an initial rapid association equilibrium followed by a transition to a second bound state prior to catalysis. Furthermore, the ligase binds and dissociates from nicked and nonsubstrate dsDNA rapidly with initial association affinities on the order of 100 nM regardless of enzyme adenylylation state. DNA binding occurs through a two-step mechanism in all cases, confirming prior proposals of transient binding followed by a transition to a productive ligase·nDNA (Lig·nDNA) conformation but suggesting that weaker nonproductive "closed" complexes are formed as well. These observations demonstrate the mechanistic underpinnings of competitive inhibition by rapid binding of nonsubstrate DNA, and of substrate inhibition by blocking of the self-adenylylation reaction through nick binding by deadenylylated ligase. Our analysis further reveals that product release is not the rate-determining step in turnover.

  13. Up-regulation of WRN and DNA ligase IIIalpha in chronic myeloid leukemia: consequences for the repair of DNA double-strand breaks.

    PubMed

    Sallmyr, Annahita; Tomkinson, Alan E; Rassool, Feyruz V

    2008-08-15

    Expression of oncogenic BCR-ABL in chronic myeloid leukemia (CML) results in increased reactive oxygen species (ROS) that in turn cause increased DNA damage, including DNA double-strand breaks (DSBs). We have previously shown increased error-prone repair of DSBs by nonhomologous end-joining (NHEJ) in CML cells. Recent reports have identified alternative NHEJ pathways that are highly error prone, prompting us to examine the role of the alternative NHEJ pathways in BCR-ABL-positive CML. Importantly, we show that key proteins in the major NHEJ pathway, Artemis and DNA ligase IV, are down-regulated, whereas DNA ligase IIIalpha, and the protein deleted in Werner syndrome, WRN, are up-regulated. DNA ligase IIIalpha and WRN form a complex that is recruited to DSBs in CML cells. Furthermore, "knockdown" of either DNA ligase IIIalpha or WRN leads to increased accumulation of unrepaired DSBs, demonstrating that they contribute to the repair of DSBs. These results indicate that altered DSB repair in CML cells is caused by the increased activity of an alternative NHEJ repair pathway, involving DNA ligase IIIalpha and WRN. We suggest that, although the repair of ROS-induced DSBs by this pathway contributes to the survival of CML cells, the resultant genomic instability drives disease progression.

  14. Tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can specifically target bacterial DNA ligases and can distinguish them from human DNA ligase I.

    PubMed

    Yadav, Nisha; Khanam, Taran; Shukla, Ankita; Rai, Niyati; Hajela, Kanchan; Ramachandran, Ravishankar

    2015-05-21

    DNA ligases are critical components for DNA metabolism in all organisms. NAD(+)-dependent DNA ligases (LigA) found exclusively in bacteria and certain entomopoxviruses are drawing increasing attention as therapeutic targets as they differ in their cofactor requirement from ATP-dependent eukaryotic homologs. Due to the similarities in the cofactor binding sites of the two classes of DNA ligases, it is necessary to find determinants that can distinguish between them for the exploitation of LigA as an anti-bacterial target. In the present endeavour, we have synthesized and evaluated a series of tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives for their ability to distinguish between bacterial and human DNA ligases. The in vivo inhibition assays that employed LigA deficient E. coli GR501 and S. typhimurium LT2 bacterial strains, rescued by ATP-dependent T4 DNA ligase or Mycobacterium tuberculosis NAD(+)-dependent DNA ligase (Mtb LigA), respectively, showed that the compounds can specifically inhibit bacterial LigA. The in vitro enzyme inhibition assays using purified MtbLigA, human DNA ligase I & T4 DNA ligase showed specific inhibition of MtbLigA at low micromolar range. Our results demonstrate that tricyclic dihydrobenzoxazepine and tetracyclic indole derivatives can distinguish between bacterial and human DNA ligases by ∼5-folds. In silico docking and enzyme inhibition assays identified that the compounds bind to the cofactor binding site and compete with the cofactor. Ethidium bromide displacement and gel-shift assays showed that the inhibitors do not exhibit any unwanted general interactions with the substrate DNA. These results set the stage for the detailed exploration of this compound class for development as antibacterials.

  15. From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

    PubMed Central

    Tanabe, Maiko; Nishida, Hirokazu

    2015-01-01

    DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures. PMID:26508902

  16. From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase.

    PubMed

    Tanabe, Maiko; Ishino, Yoshizumi; Nishida, Hirokazu

    2015-01-01

    DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures.

  17. Influence of polyethylene glycol on the ligation reaction with calf thymus DNA ligases I and II.

    PubMed

    Teraoka, H; Tsukada, K

    1987-01-01

    High concentrations of the nonspecific macromolecule polyethylene glycol 6000 (PEG 6000) enabled DNA ligases I and II from calf thymus to catalyze intermolecular blunt-end ligation of duplex DNA. Intermolecular cohesive-end ligation with these enzymes was markedly stimulated in the presence of 10-16% (w/v) PEG 6000. The effect of PEG 6000 (4-16%) on the sealing of single-stranded breaks in duplex DNA with DNA ligases I and II was not appreciably stimulatory but rather inhibitory. PEG 6000 (15%) enhanced more twofold the rate of DNA ligase II-AMP complex formation, but moderately suppressed the rate of formation of DNA ligase 1-AMP complex. Polyamines and KCl inhibited blunt-end and cohesive-end ligations with DNA ligases I and II in the presence of PEG 6000.

  18. Structure of the adenylation domain of NAD(+)-dependent DNA ligase from Staphylococcus aureus.

    PubMed

    Han, Seungil; Chang, Jeanne S; Griffor, Matt

    2009-11-01

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD(+)-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD(+)-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD(+)-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  19. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  20. Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus.

    PubMed

    Lu, Jing; Tong, Jie; Feng, Hong; Huang, Jianmin; Afonso, Claudio L; Rock, Dan L; Barany, Francis; Cao, Weiguo

    2004-09-01

    The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+). MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

  1. ATM mediates oxidative stress-induced dephosphorylation of DNA ligase IIIalpha.

    PubMed

    Dong, Zhiwan; Tomkinson, Alan E

    2006-01-01

    Among the three mammalian genes encoding DNA ligases, only the LIG3 gene does not have a homolog in lower eukaryotes. In somatic mammalian cells, the nuclear form of DNA ligase IIIalpha forms a stable complex with the DNA repair protein XRCC1 that is also found only in higher eukaryotes. Recent studies have shown that XRCC1 participates in S phase-specific DNA repair pathways independently of DNA ligase IIIalpha and is constitutively phosphorylated by casein kinase II. In this study we demonstrate that DNA ligase IIIalpha, unlike XRCC1, is phosphorylated in a cell cycle-dependent manner. Specifically, DNA ligase IIIalpha is phosphorylated on Ser123 by the cell division cycle kinase Cdk2 beginning early in S phase and continuing into M phase. Interestingly, treatment of S phase cells with agents that cause oxygen free radicals induces the dephosphorylation of DNA ligase IIIalpha. This oxidative stress-induced dephosphorylation of DNA ligase IIIalpha is dependent upon the ATM (ataxia-telangiectasia mutated) kinase and appears to involve inhibition of Cdk2 and probably activation of a phosphatase.

  2. ATM mediates oxidative stress-induced dephosphorylation of DNA ligase IIIα

    PubMed Central

    Dong, Zhiwan; Tomkinson, Alan E.

    2006-01-01

    Among the three mammalian genes encoding DNA ligases, only the LIG3 gene does not have a homolog in lower eukaryotes. In somatic mammalian cells, the nuclear form of DNA ligase IIIα forms a stable complex with the DNA repair protein XRCC1 that is also found only in higher eukaryotes. Recent studies have shown that XRCC1 participates in S phase-specific DNA repair pathways independently of DNA ligase IIIα and is constitutively phosphorylated by casein kinase II. In this study we demonstrate that DNA ligase IIIα, unlike XRCC1, is phosphorylated in a cell cycle-dependent manner. Specifically, DNA ligase IIIα is phosphorylated on Ser123 by the cell division cycle kinase Cdk2 beginning early in S phase and continuing into M phase. Interestingly, treatment of S phase cells with agents that cause oxygen free radicals induces the dephosphorylation of DNA ligase IIIα. This oxidative stress-induced dephosphorylation of DNA ligase IIIα is dependent upon the ATM (ataxia-telangiectasia mutated) kinase and appears to involve inhibition of Cdk2 and probably activation of a phosphatase. PMID:17040896

  3. DNA-ligase activities appear normal in the CHO mutant EM9.

    PubMed

    Chan, J Y; Thompson, L H; Becker, F F

    1984-01-01

    The Chinese hamster ovary (CHO) mutant strain EM9 was previously shown to be hypersensitive to killing by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS), to have a 12-fold increased baseline incidence of sister-chromatid exchanges (SCE), and to be defective in rejoining DNA strand breaks after treatment with EMS, MMS, or X-rays. A study was performed to determine if the primary biochemical defect might be a DNA ligase. DNA-ligase activities were assayed and compared after separation of the multiple forms of ligase by AcA 34 gel-filtration chromatography of total cellular extracts. In EM9 cells the levels of the presumptive replicative forms, DNA ligase Ia (480 kd) and ligase Ib (240 kd) were about 50% and 60%, respectively, of those in the parental AA8 cells, whereas DNA ligase II (80 kd) was unaltered in EM9 . In a phenotypic revertant line ( 9R1 ) ligases Ia, Ib and II levels were 35%, 37% and 100%, respectively, of those in AA8 . The reduced levels of ligases Ia and Ib in EM9 and 9R1 cells are apparently not related directly to the mutant phenotype and may be attributable to the somewhat slower growth rates of these strains compared with those of AA8 . To determine if the repair defect in EM9 might reside in the ability to induce DNA-ligase activity after treatment with a DNA-damaging agent, AA8 and EM9 cells were treated with MMS at 30 micrograms/ml for 60 min before preparing fractions for ligase assays. Under these conditions the activities of ligases Ia and Ib decreases 70-80% in both cell lines, but ligase II increased 2.0- and 2.6-fold, respectively, in AA8 and EM9 . As a further test of defective ligase activities in EM9 , assays were performed in the presence of 0.1 M NaCl or after heating the fractions for 10 min at 50 degrees C. Although all 3 forms of ligase showed altered activity under both of these conditions, there were no significant differences between EM9 and AA8 cells. These data combined with the above results provide strong

  4. Escherichia coli DNA ligase B may mitigate damage from oxidative stress.

    PubMed

    Bodine, Truston J; Evangelista, Michael A; Chang, Huan Ting; Ayoub, Christopher A; Samuel, Buck S; Sucgang, Richard; Zechiedrich, Lynn

    2017-01-01

    Escherichia coli encodes two DNA ligases, ligase A, which is essential under normal laboratory growth conditions, and ligase B, which is not. Here we report potential functions of ligase B. We found that across the entire Enterobacteriaceae family, ligase B is highly conserved in both amino acid identity and synteny with genes associated with oxidative stress. Deletion of ligB sensitized E. coli to specific DNA damaging agents and antibiotics resulted in a weak mutator phenotype, and decreased biofilm formation. Overexpression of ligB caused a dramatic extension of lag phase that eventually resumed normal growth. The ligase function of ligase B was not required to mediate the extended lag phase, as overexpression of a ligase-deficient ligB mutant also blocked growth. Overexpression of ligB during logarithmic growth caused an immediate block of cell growth and DNA replication, and death of about half of cells. These data support a potential role for ligase B in the base excision repair pathway or the mismatch repair pathway.

  5. Defects in DNA ligase I trigger PCNA ubiquitylation at Lys 107.

    PubMed

    Das-Bradoo, Sapna; Nguyen, Hai Dang; Wood, Jamie L; Ricke, Robin M; Haworth, Justin C; Bielinsky, Anja-Katrin

    2010-01-01

    In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalysed by DNA ligase I (ref. 1). An individual with a DNA ligase I deficiency exhibits growth retardation, sunlight sensitivity and severe immunosuppression, probably due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. As DNA replication and DDR pathways are highly conserved in eukaryotes, we used Saccharomyces cerevisiae as a model system to address this issue. We uncovered a new pathway, which facilitates ubiquitylation at Lys 107 of proliferating cell nuclear antigen (PCNA). Unlike ubiquitylation at Lys 164 of PCNA in response to UV irradiation, which triggers translesion synthesis, modification of Lys 107 is not dependent on the ubiquitin conjugating enzyme (E2) Rad6 (ref. 4) nor the ubiquitin ligase (E3) Rad18 (ref. 5), but requires the E2 variant Mms2 (ref. 6) in conjunction with Ubc4 (ref. 7) and the E3 Rad5 (Refs 8, 9). Surprisingly, DNA ligase I-deficient S. cerevisiae cdc9-1 cells that carry a PCNAK107R mutation are inviable, because they cannot activate a robust DDR. Furthermore, we show that ubiquitylation of PCNA in response to DNA ligase I deficiency is conserved in humans, yet the lysine residue that is modified remains to be determined. We propose that PCNA ubiquitylation provides a 'DNA damage code' that allows cells to categorize different types of defects that arise during DNA replication.

  6. Structural Basis for Nick Recognition by a Minimal Pluripotent DNA Ligase

    SciTech Connect

    Nair,P.; Nandakumar, J.; Smith, P.; Odell, M.; Lima, C.; Shuman, S.

    2007-01-01

    Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-{angstrom} crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO{sub 4} nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a {beta}-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO{sub 4} termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.

  7. Effects of DNA-binding drugs on T4 DNA ligase.

    PubMed Central

    Montecucco, A; Pedrali-Noy, G; Spadari, S; Lestingi, M; Ciarrocchi, G

    1990-01-01

    A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate. Images Fig. 2. Fig. 4. Fig. 5. PMID:2156493

  8. A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase.

    PubMed

    Shapiro, Adam B; Eakin, Ann E; Walkup, Grant K; Rivin, Olga

    2011-06-01

    DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

  9. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    PubMed

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  10. Human Mre11/human Rad50/Nbs1 and DNA ligase IIIalpha/XRCC1 protein complexes act together in an alternative nonhomologous end joining pathway.

    PubMed

    Della-Maria, Julie; Zhou, Yi; Tsai, Miaw-Sheue; Kuhnlein, Jeff; Carney, James P; Paull, Tanya T; Tomkinson, Alan E

    2011-09-30

    Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

  11. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    PubMed

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Identification and Validation of Human DNA Ligase Inhibitors Using Computer-Aided Drug Design

    PubMed Central

    Zhong, Shijun; Chen, Xi; Zhu, Xiao; Dziegielewska, Barbara; Bachman, Kurtis E.; Ellenberger, Tom; Ballin, Jeff D.; Wilson, Gerald M.; Tomkinson, Alan E.; MacKerell, Alexander D.

    2009-01-01

    Linking together of DNA strands by DNA ligases is essential for DNA replication and repair. Since many therapies used to treat cancer act by causing DNA damage, there is growing interest in the development of DNA repair inhibitors. Accordingly, virtual database screening and experimental evaluation were applied to identify inhibitors of human DNA ligase I (hLigI). When a DNA binding site within the DNA binding domain (DBD) of hLigI was targeted, more than 1 million compounds were screened from which 192 were chosen for experimental evaluation. In DNA joining assays, 10 compounds specifically inhibited hLigI, 5 of which also inhibited the proliferation of cultured human cell lines. Analysis of the 10 active compounds revealed the utility of including multiple protein conformations and chemical clustering in the virtual screening procedure. The identified ligase inhibitors are structurally diverse and have druglike physical and molecular characteristics making them ideal for further drug development studies. PMID:18630893

  13. Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.

    PubMed

    Barany, F; Gelfand, D H

    1991-12-20

    Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.

  14. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    PubMed

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  15. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

  16. Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway.

    PubMed

    He, Yi; Shao, Yanchun; Chen, Fusheng

    2014-01-01

    Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (MrΔlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the MrΔlig4 strain increased four-fold compared with that in the wild-type strain, reached 68 % and 85 %, respectively. Thus, the MrΔlig4 strain is a promising host for efficient genetic manipulation. In addition, the MrΔlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate.

  17. DNA Ligase I is an In Vivo Substrate of DNA-Dependent Protein Kinase and is Activated by Phosphorylation in Response to DNA Double-Strand Breaks

    DTIC Science & Technology

    2006-01-01

    anlysis. to the procedure described by Malanga and Althaus (8). Gel Electrophoresis and A utoradiography. Immunopre- DNA Ligase and Protein Assays. DNA...by casein kinase 11, EMBO J. 11, 2925-2933. In conclusion, we have demonstrated that DNA ligase I 8. Malanga , M., and Althaus, F. R. (1994) Poly (ADP

  18. Cloning, molecular characterization and expression of a DNA-ligase from a new bacteriophage: Phax1.

    PubMed

    Setayesh, Neda; Sabouri-Shahrbabak, Saleheh; Bakherad, Hamid; Sepehrizadeh, Zargham

    2013-12-01

    DNA ligases join 3' hydroxyl and 5' phosphate ends in double stranded DNA and are necessary for maintaining the integrity of genome. The gene encoding a new Escherichia phage (Phax1) DNA ligase was cloned and sequenced. The gene contains an open reading frame with 1,428 base pairs, encoding 475 amino acid residues. Alignment of the entire amino acid sequence showed that Phax1 DNA ligase has a high degree of sequence homology with ligases from Escherichia (vB_EcoM_CBA120), Salmonella (PhiSH19 and SFP10), Shigella (phiSboM-AG3), and Deftia (phiW-14) phages. The Phax1 DNA ligase gene was expressed under the control of the T7lac promoter on the pET-16b (+) in Escherichia coli Rossetta gami. The enzyme was then homogeneously purified by a metal affinity column. Enzymatic activity of the recombinant DNA ligase was assayed by an in-house PCR-based method.

  19. Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida.

    PubMed

    Williamson, Adele; Pedersen, Hege

    2014-05-01

    The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATP-dependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an Escherichia coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-S-transferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents. As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active A. salmonicida ATP-dependent DNA ligase.

  20. Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand

    PubMed Central

    Patel, Maha P.; Baum, Dana A.; Silverman, Scott K.

    2008-01-01

    DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates. PMID:18022669

  1. Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells.

    PubMed

    Ruhanen, Heini; Ushakov, Kathy; Yasukawa, Takehiro

    2011-12-01

    Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.

  2. Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

    PubMed

    Pergolizzi, Giulia; Cominetti, Marco M D; Butt, Julea N; Field, Robert A; Bowater, Richard P; Wagner, Gerd K

    2015-06-14

    We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of β-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of β-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in β-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

  3. Inhibition of human DNA ligase I activity by zinc and cadmium and the fidelity of ligation.

    PubMed

    Yang, S W; Becker, F F; Chan, J Y

    1996-01-01

    Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.

  4. Enzyme-regulated activation of DNAzyme: a novel strategy for a label-free colorimetric DNA ligase assay and ligase-based biosensing.

    PubMed

    He, Kaiyu; Li, Wang; Nie, Zhou; Huang, Yan; Liu, Zhuoliang; Nie, Lihua; Yao, Shouzhuo

    2012-03-26

    The DNA nick repair catalyzed by DNA ligase is significant for fundamental life processes, such as the replication, repair, and recombination of nucleic acids. Here, we have employed ligase to regulate DNAzyme activity and developed a homogeneous, colorimetric, label-free and DNAzyme-based strategy to detect DNA ligase activity. This novel strategy relies on the ligation-trigged activation or production of horseradish peroxidase mimicking DNAzyme that catalyzes the generation of a color change signal; this results in a colorimetric assay of DNA ligase activity. Using T4 DNA ligase as a model, we have proposed two approaches to demonstrate the validity of the DNAzyme strategy. The first approach utilizes an allosteric hairpin-DNAzyme probe specifically responsive to DNA ligation; this approach has a wide detection range from 0.2 to 40 U mL(-1) and a detection limit of 0.2 U mL(-1). Furthermore, the approach was adapted to probe nucleic acid phosphorylation and single nucleotide mismatch. The second approach employs a "split DNA machine" to produce numerous DNAzymes after being reassembled by DNA ligase; this greatly enhances the detection sensitivity by a signal amplification cascade to achieve a detection limit of 0.01 U mL(-1).

  5. Evolution of DNA ligases of Nucleo-Cytoplasmic Large DNA viruses of eukaryotes: a case of hidden complexity

    PubMed Central

    2009-01-01

    Background Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood. Results Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III. Conclusions Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different

  6. Discovery and design of DNA and RNA ligase inhibitors in infectious microorganisms

    PubMed Central

    Swift, Robert V.; Amaro, Rommie E.

    2009-01-01

    Background Members of the nucleotidyltransferase superfamily known as DNA and RNA ligases carry out the enzymatic process of polynucleotide ligation. These guardians of genomic integrity share a three-step ligation mechanism, as well as common core structural elements. Both DNA and RNA ligases have experienced a surge of recent interest as chemotherapeutic targets for the treatment of a range of diseases, including bacterial infection, cancer, and the diseases caused by the protozoan parasites known as trypanosomes. Objective In this review, we will focus on efforts targeting pathogenic microorganisms; specifically, bacterial NAD+-dependent DNA ligases, which are promising broad-spectrum antibiotic targets, and ATP-dependent RNA editing ligases from Trypanosoma brucei, the species responsible for the devastating neurodegenerative disease, African sleeping sickness. Conclusion High quality crystal structures of both NAD+-dependent DNA ligase and the Trypanosoma brucei RNA editing ligase have facilitated the development of a number of promising leads. For both targets, further progress will require surmounting permeability issues and improving selectivity and affinity. PMID:20354588

  7. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    PubMed

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  8. Biochemical and structural characterization of DNA ligases from bacteria and archaea

    PubMed Central

    Pergolizzi, Giulia; Wagner, Gerd K.; Bowater, Richard P.

    2016-01-01

    DNA ligases are enzymes that seal breaks in the backbones of DNA, leading to them being essential for the survival of all organisms. DNA ligases have been studied from many different types of cells and organisms and shown to have diverse sizes and sequences, with well conserved specific sequences that are required for enzymatic activity. A significant number of DNA ligases have been isolated or prepared in recombinant forms and, here, we review their biochemical and structural characterization. All DNA ligases contain an essential lysine that transfers an adenylate group from a co-factor to the 5′-phosphate of the DNA end that will ultimately be joined to the 3′-hydroxyl of the neighbouring DNA strand. The essential DNA ligases in bacteria use β-nicotinamide adenine dinucleotide (β-NAD+) as their co-factor whereas those that are essential in other cells use adenosine-5′-triphosphate (ATP) as their co-factor. This observation suggests that the essential bacterial enzyme could be targeted by novel antibiotics and the complex molecular structure of β-NAD+ affords multiple opportunities for chemical modification. Several recent studies have synthesized novel derivatives and their biological activity against a range of DNA ligases has been evaluated as inhibitors for drug discovery and/or non-natural substrates for biochemical applications. Here, we review the recent advances that herald new opportunities to alter the biochemical activities of these important enzymes. The recent development of modified derivatives of nucleotides highlights that the continued combination of structural, biochemical and biophysical techniques will be useful in targeting these essential cellular enzymes. PMID:27582505

  9. Extreme Growth Failure is a Common Presentation of Ligase IV Deficiency

    PubMed Central

    Murray, Jennie E; Bicknell, Louise S; Yigit, Gökhan; Duker, Angela L; van Kogelenberg, Margriet; Haghayegh, Sara; Wieczorek, Dagmar; Kayserili, Hülya; Albert, Michael H; Wise, Carol A; Brandon, January; Kleefstra, Tjitske; Warris, Adilia; van der Flier, Michiel; Bamforth, J Steven; Doonanco, Kurston; Adès, Lesley; Ma, Alan; Field, Michael; Johnson, Diana; Shackley, Fiona; Firth, Helen; Woods, C Geoffrey; Nürnberg, Peter; Gatti, Richard A; Hurles, Matthew; Bober, Michael B; Wollnik, Bernd; Jackson, Andrew P

    2014-01-01

    Ligase IV syndrome is a rare differential diagnosis for Nijmegen breakage syndrome owing to a shared predisposition to lympho-reticular malignancies, significant microcephaly, and radiation hypersensitivity. Only 16 cases with mutations in LIG4 have been described to date with phenotypes varying from malignancy in developmentally normal individuals, to severe combined immunodeficiency and early mortality. Here, we report the identification of biallelic truncating LIG4 mutations in 11 patients with microcephalic primordial dwarfism presenting with restricted prenatal growth and extreme postnatal global growth failure (average OFC −10.1 s.d., height −5.1 s.d.). Subsequently, most patients developed thrombocytopenia and leucopenia later in childhood and many were found to have previously unrecognized immunodeficiency following molecular diagnosis. None have yet developed malignancy, though all patients tested had cellular radiosensitivity. A genotype–phenotype correlation was also noted with position of truncating mutations corresponding to disease severity. This work extends the phenotypic spectrum associated with LIG4 mutations, establishing that extreme growth retardation with microcephaly is a common presentation of bilallelic truncating mutations. Such growth failure is therefore sufficient to consider a diagnosis of LIG4 deficiency and early recognition of such cases is important as bone marrow failure, immunodeficiency, and sometimes malignancy are long term sequelae of this disorder. PMID:24123394

  10. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori.

    PubMed

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; Kumari, Nilima; Gourinath, Samudrala

    2016-08-08

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp.

  11. Structural insight into β-Clamp and its interaction with DNA Ligase in Helicobacter pylori

    PubMed Central

    Pandey, Preeti; Tarique, Khaja Faisal; Mazumder, Mohit; Rehman, Syed Arif Abdul; kumari, Nilima; Gourinath, Samudrala

    2016-01-01

    Helicobacter pylori, a gram-negative and microaerophilic bacterium, is the major cause of chronic gastritis, gastric ulcers and gastric cancer. Owing to its central role, DNA replication machinery has emerged as a prime target for the development of antimicrobial drugs. Here, we report 2Å structure of β-clamp from H. pylori (Hpβ-clamp), which is one of the critical components of DNA polymerase III. Despite of similarity in the overall fold of eubacterial β-clamp structures, some distinct features in DNA interacting loops exists that have not been reported previously. The in silico prediction identified the potential binders of β-clamp such as alpha subunit of DNA pol III and DNA ligase with identification of β-clamp binding regions in them and validated by SPR studies. Hpβ-clamp interacts with DNA ligase in micromolar binding affinity. Moreover, we have successfully determined the co-crystal structure of β-clamp with peptide from DNA ligase (not reported earlier in prokaryotes) revealing the region from ligase that interacts with β-clamp. PMID:27499105

  12. Broad nucleotide cofactor specificity of DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus and its evolutionary significance.

    PubMed

    Kim, Jun-Hwan; Lee, Kang-Keun; Sun, Younguk; Seo, Gang-Jin; Cho, Sung Suk; Kwon, Suk Hyung; Kwon, Suk-Tae

    2013-05-01

    The nucleotide cofactor specificity of the DNA ligase from the hyperthermophilic crenarchaeon Hyperthermus butylicus (Hbu) was studied to investigate the evolutionary relationship of DNA ligases. The Hbu DNA ligase gene was expressed under control of the T7lac promoter of pTARG in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified using the IMPACT™-CN system (intein-mediated purification with an affinity chitin-binding tag) and cation-ion (Arg-tag) chromatography. The optimal temperature for Hbu DNA ligase activity was 75 °C, and the optimal pH was 8.0 in Tris-HCl. The activity was highly dependent on MgCl2 or MnCl2 with maximal activity above 5 mM MgCl2 and 2 mM MnCl2. Notably, Hbu DNA ligase can use ADP and GTP in addition to ATP. The broad nucleotide cofactor specificity of Hbu DNA ligase might exemplify an undifferentiated ancestral stage in the evolution of DNA ligases. This study provides new evidence for possible evolutionary relationships among DNA ligases.

  13. Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells.

    PubMed

    Le Chalony, Catherine; Hoffschir, Françoise; Gauthier, Laurent R; Gross, Julia; Biard, Denis S; Boussin, François D; Pennaneach, Vincent

    2012-09-01

    DNA ligase I (LigI) plays a central role in the joining of strand interruptions during replication and repair. In our current study, we provide evidence that DNA ligase III (LigIII) and XRCC1, which form a complex that functions in single-strand break repair, are required for the proliferation of mammalian LigI-depleted cells. We show from our data that in cells with either dysfunctional LigI activity or depleted of this enzyme, both LigIII and XRCC1 are retained on the chromatin and accumulate at replication foci. We also demonstrate that the LigI and LigIII proteins cooperate to inhibit sister chromatid exchanges but that only LigI prevents telomere sister fusions. Taken together, these results suggest that in cells with dysfunctional LigI, LigIII contributes to the ligation of replication intermediates but not to the prevention of telomeric instability.

  14. ZRF1 mediates remodeling of E3 ligases at DNA lesion sites during nucleotide excision repair

    PubMed Central

    Gracheva, Ekaterina; Chitale, Shalaka; Wilhelm, Thomas; Rapp, Alexander; Byrne, Jonathan; Stadler, Jens; Medina, Rebeca; Cardoso, M. Cristina

    2016-01-01

    Faithful DNA repair is essential to maintain genome integrity. Ultraviolet (UV) irradiation elicits both the recruitment of DNA repair factors and the deposition of histone marks such as monoubiquitylation of histone H2A at lesion sites. Here, we report how a ubiquitin E3 ligase complex specific to DNA repair is remodeled at lesion sites in the global genome nucleotide excision repair (GG-NER) pathway. Monoubiquitylation of histone H2A (H2A-ubiquitin) is catalyzed predominantly by a novel E3 ligase complex consisting of DDB2, DDB1, CUL4B, and RING1B (UV–RING1B complex) that acts early during lesion recognition. The H2A-ubiquitin binding protein ZRF1 mediates remodeling of this E3 ligase complex directly at the DNA lesion site, causing the assembly of the UV–DDB–CUL4A E3 ligase complex (DDB1–DDB2–CUL4A-RBX1). ZRF1 is an essential factor in GG-NER, and its function at damaged chromatin sites is linked to damage recognition factor XPC. Overall, the results shed light on the interplay between epigenetic and DNA repair recognition factors at DNA lesion sites. PMID:27091446

  15. Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

    PubMed Central

    Zhelkovsky, Alexander M.; McReynolds, Larry A.

    2014-01-01

    RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3′p substrate to generate an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA3′pp5′A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA 3′pp5′A, the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3′p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in double-stranded DNA to produce a 3′-adenylated product. These 3′-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates. PMID:25324547

  16. Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation.

    PubMed

    Bhat, K Ramachandra; Benton, Betty J; Ray, Radharaman

    2006-01-01

    Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.

  17. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

    PubMed

    Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

    2016-02-02

    Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

  18. Effects of 2'-O-methyl nucleotide on ligation capability of T4 DNA ligase.

    PubMed

    Zhao, Bin; Tong, Zhaoxue; Zhao, Guojie; Mu, Runqing; Shang, Hong; Guan, Yifu

    2014-09-01

    To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.

  19. A high-throughput assay for the comprehensive profiling of DNA ligase fidelity.

    PubMed

    Lohman, Gregory J S; Bauer, Robert J; Nichols, Nicole M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Evans, Thomas C

    2016-01-29

    DNA ligases have broad application in molecular biology, from traditional cloning methods to modern synthetic biology and molecular diagnostics protocols. Ligation-based detection of polynucleotide sequences can be achieved by the ligation of probe oligonucleotides when annealed to a complementary target sequence. In order to achieve a high sensitivity and low background, the ligase must efficiently join correctly base-paired substrates, while discriminating against the ligation of substrates containing even one mismatched base pair. In the current study, we report the use of capillary electrophoresis to rapidly generate mismatch fidelity profiles that interrogate all 256 possible base-pair combinations at a ligation junction in a single experiment. Rapid screening of ligase fidelity in a 96-well plate format has allowed the study of ligase fidelity in unprecedented depth. As an example of this new method, herein we report the ligation fidelity of Thermus thermophilus DNA ligase at a range of temperatures, buffer pH and monovalent cation strength. This screen allows the selection of reaction conditions that maximize fidelity without sacrificing activity, while generating a profile of specific mismatches that ligate detectably under each set of conditions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Use of adenylate kinase as a solubility tag for high level expression of T4 DNA ligase in Escherichia coli.

    PubMed

    Liu, Xinxin; Huang, Anliang; Luo, Dan; Liu, Haipeng; Han, Huzi; Xu, Yang; Liang, Peng

    2015-05-01

    The discovery of T4 DNA ligase in 1960s was pivotal in the spread of molecular biotechnology. The enzyme has become ubiquitous for recombinant DNA routinely practiced in biomedical research around the globe. Great efforts have been made to express and purify T4 DNA ligase to meet the world demand, yet over-expression of soluble T4 DNA ligase in E. coli has been difficult. Here we explore the use of adenylate kinase to enhance T4 DNA ligase expression and its downstream purification. E.coli adenylate kinase, which can be expressed in active form at high level, was fused to the N-terminus of T4 DNA ligase. The resulting His-tagged AK-T4 DNA ligase fusion protein was greatly over-expressed in E. coli, and readily purified to near homogeneity via two purification steps consisting of Blue Sepharose and Ni-NTA chromatography. The purified AK-T4 DNA ligase not only is fully active for DNA ligation, but also can use ADP in addition to ATP as energy source since adenylate kinase converts ADP to ATP and AMP. Thus adenylate kinase may be used as a solubility tag to facilitate recombinant protein expression as well as their downstream purification. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

    PubMed

    Günther Sillero, María A; de Diego, Anabel; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2008-05-15

    T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

  2. Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

    PubMed

    Stokes, Suzanne S; Huynh, Hoan; Gowravaram, Madhusudhan; Albert, Robert; Cavero-Tomas, Marta; Chen, Brendan; Harang, Jenna; Loch, James T; Lu, Min; Mullen, George B; Zhao, Shannon; Liu, Ce-Feng; Mills, Scott D

    2011-08-01

    Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

  3. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  4. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  5. Effective interaction studies for inhibition of DNA ligase protein from Staphylococcus aureus.

    PubMed

    Vijayalakshmi, Periyasamy; Daisy, Pitchai

    2015-02-01

    Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. It is among the most important causative agent of human infections in the twenty-first century. DNA ligase is the main protein responsible for the replication of S. aureus. In order to control the replication mechanism, DNA ligase is a successive drug target, hence we have chosen this protein for this study. We performed virtual screening using ZINC database for identification of potent inhibitor against DNA ligase. Based on the scoring methods, we have selected best five compounds from the ZINC database. In order to improve the accuracy, selected compounds were subjected into Quantum Polarized Ligand Docking (QPLD) docking, for which the results showed high docking score, compared to glide docking score. QPLD is more accurate as it includes charges in the scoring function, which was not available in the glide docking. Binding energy calculation results also indicated that selected compounds have good binding capacity with the target protein. In addition, these compounds on screening have good absorption, distribution, metabolism, excretion and toxicity property. In this study, we identified few compounds that particularly work against DNA ligase protein, having better interaction phenomenon and it would help further the experimental analysis.

  6. An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination.

    PubMed Central

    Mackey, Z B; Ramos, W; Levin, D S; Walter, C A; McCarrey, J R; Tomkinson, A E

    1997-01-01

    Three mammalian genes encoding DNA ligases have been identified. However, the role of each of these enzymes in mammalian DNA metabolism has not been established. In this study, we show that two forms of mammalian DNA ligase III, alpha and beta, are produced by a conserved tissue-specific alternative splicing mechanism involving exons encoding the C termini of the polypeptides. DNA ligase III-alpha cDNA, which encodes a 103-kDa polypeptide, is expressed in all tissues and cells, whereas DNA ligase III-beta cDNA, which encodes a 96-kDa polypeptide, is expressed only in the testis. During male germ cell differentiation, elevated expression of DNA ligase III-beta mRNA is restricted, beginning only in the latter stages of meiotic prophase and ending in the round spermatid stage. In 96-kDa DNA ligase III-beta, the C-terminal 77 amino acids of DNA ligase III-alpha are replaced by a different 17- to 18-amino acid sequence. As reported previously, the 103-kDa DNA ligase III-alpha interacts with the DNA strand break repair protein encoded by the human XRCC1 gene. In contrast, the 96-kDa DNA ligase III-beta does not interact with XRCC1, indicating that DNA ligase III-beta may play a role in cellular functions distinct from the DNA repair pathways involving the DNA ligase III-alpha x XRCC1 complex. The distinct biochemical properties of DNA ligase III-beta, in combination with the tissue- and cell-type-specific expression of DNA ligase III-beta mRNA, suggest that this form of DNA ligase III is specifically involved in the completion of homologous recombination events that occur during meiotic prophase. PMID:9001252

  7. Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

  8. Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

  9. Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.

    PubMed

    Schneider, Nils; Meier, Matthias

    2017-02-01

    Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications. © 2017 Schneider and Meier; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. A plant DNA ligase is an important determinant of seed longevity.

    PubMed

    Waterworth, Wanda M; Masnavi, Ghzaleh; Bhardwaj, Rajni M; Jiang, Qing; Bray, Clifford M; West, Christopher E

    2010-09-01

    DNA repair is important for maintaining genome integrity. In plants, DNA damage accumulated in the embryo of seeds is repaired early in imbibition, and is important for germination performance and seed longevity. An essential step in most repair pathways is the DNA ligase-mediated rejoining of single- and double-strand breaks. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Here, we report the characterization of DNA LIGASE VI, which is only found in plant species. The primary structure of this ligase shows a unique N-terminal region that contains a β-CASP motif, which is found in a number of repair proteins, including the DNA double-strand break (DSB) repair factor Artemis. Phenotypic analysis revealed a delay in the germination of atlig6 mutants compared with wild-type lines, and this delay becomes markedly exacerbated in the presence of the genotoxin menadione. Arabidopsis atlig6 and atlig6 atlig4 mutants display significant hypersensitivity to controlled seed ageing, resulting in delayed germination and reduced seed viability relative to wild-type lines. In addition, atlig6 and atlig6 atlig4 mutants display increased sensitivity to low-temperature stress, resulting in delayed germination and reduced seedling vigour upon transfer to standard growth conditions. Seeds display a rapid transcriptional DNA DSB response, which is activated in the earliest stages of water imbibition, providing evidence for the accumulation of cytotoxic DSBs in the quiescent seed. These results implicate AtLIG6 and AtLIG4 as major determinants of Arabidopsis seed quality and longevity.

  11. Mitochondrial DNA ligase is dispensable for the viability of cultured cells but essential for mtDNA maintenance.

    PubMed

    Shokolenko, Inna N; Fayzulin, Rafik Z; Katyal, Sachin; McKinnon, Peter J; Wilson, Glenn L; Alexeyev, Mikhail F

    2013-09-13

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ(0) phenotype.

  12. Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance*

    PubMed Central

    Shokolenko, Inna N.; Fayzulin, Rafik Z.; Katyal, Sachin; McKinnon, Peter J.; Wilson, Glenn L.; Alexeyev, Mikhail F.

    2013-01-01

    Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ0 phenotype. PMID:23884459

  13. Novel method for screening of radioprotective agents providing protection to DNA ligase against gamma radiation induced damage.

    PubMed

    Mishra, Saurabh; Malhotra, Poonam; Gupta, Ashutosh K; Singh, Praveen K; Mishra, Anil K; Javed, Saleem; Kumar, Raj

    2014-02-01

    A simple, sensitive and novel method was developed to screen out potential agents able to protect functional activity of DNA ligase against gamma irradiation-induced damage. Repeatability, authenticity and sensitivity of the method was verified by analyzing DNA ligase protecting activities of well-known radioprotectors such as amifostine, trolox, melatonin, semiquinone glucoside derivative (SQGD) and an antioxidant gallic acid in extremely low concentration (1 μg/reaction). Two different sets (Set A and B) of T4 DNA ligase (1 unit/set) were prepared. Set 'A' (negative control) was exposed to different doses (3-5 kGy) of gamma radiation in the absence of radioprotective compounds. Set B (test) was exposed to similar doses of gamma radiation in the presence of radioprotective compounds. Following irradiation, DNA ligase was mixed with λ DNA (250 ng) pre-digested with Hind III restriction endonuclease. Ligation reaction was performed in both sets simultaneously at 22°C for 20 min and reaction product was analyzed using agarose gel electrophoresis. Complete DNA ligation was observed in samples where DNA ligase was irradiated in the presence of radioprotectective compounds, i.e., amifostine, trolox, melatonin and a natural radioprotector semiquinone glucoside derivative (SQGD) individually, while, functional impairment in ligation activity of DNA ligase was evident in samples in which DNA ligase was irradiated in the absence of a radioprotective compound. The current method was able to provide significant input to screen out radioprotective compounds able to protect DNA ligase functional activity against gamma radiation-induced functional impairment.

  14. Identification and characterization of novel ligase I inhibitors.

    PubMed

    Pandey, Monica; Kumar, Sujeet; Goldsmith, Gunaseelan; Srivastava, Mrinal; Elango, Santhini; Shameem, Mohammad; Bannerjee, Dibyendu; Choudhary, Bibha; Karki, Subhas S; Raghavan, Sathees C

    2017-02-01

    The terminal step of ligation of single and/or double-strand breaks during physiological processes such as DNA replication, repair and recombination requires participation of DNA ligases in all mammals. DNA Ligase I has been well characterised to play vital roles during these processes. Considering the indispensable role of DNA Ligase I, a therapeutic strategy to impede proliferation of cancer cells is by using specific small molecule inhibitors against it. In the present study, we have designed and chemically synthesised putative DNA Ligase I inhibitors. Based on various biochemical and biophysical screening approaches, we identify two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Both the inhibitors blocked ligation of nicks on DNA in a concentration-dependent manner, when catalysed by cell-free extracts or purified Ligase I. 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. The inhibitors did not show significant affinity towards DNA Ligase III and DNA Ligase IV. Further, addition of 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 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 optimised to target cancer cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

    PubMed Central

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

  16. Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

    PubMed

    Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F

    2016-01-01

    Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

  17. Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519.

    PubMed

    Modarres, Hassan Pezeshgi; Dorokhov, Boris D; Popov, Vladimir O; Ravin, Nikolai V; Skryabin, Konstantin G; Dal Peraro, Matteo

    2015-05-19

    The physical chemical principles underlying enzymatic thermostability are keys to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also an important factor for engineering more thermoresistant enzymes to be used in the industrial setting, such as, for instance, DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, we first address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance thermostability of the enzyme through bioinformatics analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.

  18. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    PubMed Central

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  19. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans.

    PubMed

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-07-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline.

  20. The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans

    PubMed Central

    Ross, A J; Li, M; Yu, B; Gao, M X; Derry, W B

    2011-01-01

    E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline. PMID:21233842

  1. Primary structure and genetic organization of phage T4 DNA ligase.

    PubMed Central

    Armstrong, J; Brown, R S; Tsugita, A

    1983-01-01

    The primary structure of phage T4 DNA ligase has been determined by DNA sequencing of a cloned restriction fragment containing its gene, and partial amino acid sequence analysis of the protein. The molecule has a Mr of 55,230, and contains 487 amino acids. The DNA sequence may also encode all of one and parts of two other, hitherto unidentified, T4 proteins. The four genes are closely packed, with overlaps between terminator and initiator codons of adjacent genes. Potential terminator and promoter sites for transcription are located within the coding sequence of one of the genes. PMID:6314278

  2. DNA ligase-based strategy for quantifying heterogeneous DNA methylation without sequencing.

    PubMed

    Wee, Eugene J H; Rauf, Sakandar; Shiddiky, Muhammad J A; Dobrovic, Alexander; Trau, Matt

    2015-01-01

    DNA methylation is a potential source of disease biomarkers. Typically, methylation levels are measured at individual cytosine/guanine (CpG) sites or over a short region of interest. However, regions of interest often show heterogeneous methylation comprising multiple patterns of methylation (epialleles) on individual DNA strands. Heterogeneous methylation is largely ignored because digital methods are required to deconvolute these usually complex patterns of epialleles. Currently, only single-molecule approaches, such as next generation sequencing (NGS), can provide detailed epiallele information. Because NGS is not yet feasible for routine practice, we developed a single-molecule-like approach, named for epiallele quantification (EpiQ). EpiQ uses DNA ligases and the enhanced thermal instability of short (≤19 bases) mismatched DNA probes for the relative quantification of epialleles. The assay was developed using fluorescent detection on a gel and then adapted for electrochemical detection on a microfabricated device. NGS was used to validate the analytical accuracy of EpiQ. In this proof of principle study, EpiQ detected with 90%-95% specificity each of the 8 possible epialleles for a 3-CpG cluster at the promoter region of the CDKN2B (p15) tumor suppressor gene. EpiQ successfully profiled heterogeneous methylation patterns in clinically derived samples, and the results were cross-validated with NGS. EpiQ is a potential alternative tool for characterizing heterogeneous methylation, thus facilitating its use as a biomarker. EpiQ was developed on a gel-based assay but can also easily be adapted for miniaturized chip-based platforms. © 2014 American Association for Clinical Chemistry.

  3. Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme.

    PubMed

    Mottes, M; Morandi, C; Cremaschi, S; Sgaramella, V

    1977-07-01

    Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

  4. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    PubMed Central

    Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

  5. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase.

    PubMed

    Bauer, Robert J; Evans, Thomas C; Lohman, Gregory J S

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

  6. Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

    PubMed

    Stokes, Suzanne S; Gowravaram, Madhusudhan; Huynh, Hoan; Lu, Min; Mullen, George B; Chen, Brendan; Albert, Robert; O'Shea, Thomas J; Rooney, Michael T; Hu, Haiqing; Newman, Joseph V; Mills, Scott D

    2012-01-01

    Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

  7. Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus.

    PubMed

    Tong, J; Barany, F; Cao, W

    2000-03-15

    An NAD(+)-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg(2+)or Mn(2+)as the metal cofactor. Ca(2+)and Ni(2+)mainly support formation of DNA-adenylate intermediates. The catalytic cycle is characterized by a low k (cat)value of 2 min(-1)with concomitant accumulation of the DNA - adenylate intermediate when Mg(2+)is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A < or = G/C < C/G < A/T on both the 3'- and 5'-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3'-side of the nick junction. The requirement of 3' complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3'- or 5'-side of the nick. Furthermore, most of the unligatable 3' mismatched base pairs prohibit formation of the DNA-adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5'-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.

  8. Inhibiting Mitochondrial DNA Ligase IIIα Activates Caspase 1-Dependent Apoptosis in Cancer Cells.

    PubMed

    Sallmyr, Annahita; Matsumoto, Yoshihiro; Roginskaya, Vera; Van Houten, Bennett; Tomkinson, Alan E

    2016-09-15

    Elevated levels of DNA ligase IIIα (LigIIIα) have been identified as a biomarker of an alteration in DNA repair in cancer cells that confers hypersensitivity to a LigIIIα inhibitor, L67, in combination with a poly (ADP-ribose) polymerase inhibitor. Because LigIIIα functions in the nucleus and mitochondria, we examined the effect of L67 on these organelles. Here, we show that, although the DNA ligase inhibitor selectively targets mitochondria, cancer and nonmalignant cells respond differently to disruption of mitochondrial DNA metabolism. Inhibition of mitochondrial LigIIIα in cancer cells resulted in abnormal mitochondrial morphology, reduced levels of mitochondrial DNA, and increased levels of mitochondrially generated reactive oxygen species that caused nuclear DNA damage. In contrast, these effects did not occur in nonmalignant cells. Furthermore, inhibition of mitochondrial LigIIIα activated a caspase 1-dependent apoptotic pathway, which is known to be part of inflammatory responses induced by pathogenic microorganisms in cancer, but not nonmalignant cells. These results demonstrate that the disruption of mitochondrial DNA metabolism elicits different responses in nonmalignant and cancer cells and suggests that the abnormal response in cancer cells may be exploited in the development of novel therapeutic strategies that selectively target cancer cells. Cancer Res; 76(18); 5431-41. ©2016 AACR. ©2016 American Association for Cancer Research.

  9. DNA clone of human type IV collagenase

    SciTech Connect

    Goldberg, G.I.; Eisen, A.Z.

    1990-05-08

    This patent relates to type IV collagenase, herein after also referred to as gelatinase. More particularly, the patent relates to the cDNA clone representing the full size human type IV collagenase (gelatinase). Collagens constitute the most abundant proteins of the extracellular matrix (ECM) in mammalian organisms. Collagen and other macromolecules of the ECM are deposited by resident cells and organized into a three-dimensional meshwork. This ECM environment plays an essential role in guiding cell migration, and in cell-to-cell communication during morphogenetic processes. The restructuring of the ECM during remodeling occurs as a cooperative multistep process involving a localized degradation of existing macromolecules, rearrangement of the cytoskeleton, cell translocation, and deposition of new ECM components.

  10. Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase*

    PubMed Central

    Samai, Poulami; Shuman, Stewart

    2011-01-01

    Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps. PMID:21527793

  11. Interaction of the β sliding clamp with MutS, ligase, and DNA polymerase I

    PubMed Central

    López de Saro, Francisco J.; O'Donnell, Mike

    2001-01-01

    The β and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli β clamp is known to function with DNA polymerases II and V, indicating that β also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein–protein interactions with the β clamp. Thus, β interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of β interactive proteins suggests that the prokaryotic β ring participates in a wide variety of reactions beyond its role in chromosomal replication. PMID:11459978

  12. Derivatized versions of ligase enzymes for constructing DNA sequences

    DOEpatents

    Mariella, Jr., Raymond P.; Christian, Allen T.; Tucker, James D.; Dzenitis, John M.; Papavasiliou, Alexandros P.

    2006-08-15

    A method of making very long, double-stranded synthetic poly-nucleotides. A multiplicity of short oligonucleotides is provided. The short oligonucleotides are sequentially hybridized to each other. Enzymatic ligation of the oligonucleotides provides a contiguous piece of PCR-ready DNA of predetermined sequence.

  13. [The applications of thermostable ligase chain reaction in facilitating DNA recombination].

    PubMed

    Xiangda, Zhou; Xiao, Song; Cong, Huai; Haiyan, Sun; Hongyan, Chen; Daru, Lu

    2016-02-01

    The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, "Helper", to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.

  14. Detection of low abundant mutations in DNA using single-molecule FRET and ligase detection reactions

    NASA Astrophysics Data System (ADS)

    Wabuyele, Musundi B.; Farquar, Hannah; Stryjewski, Wieslaw J.; Hammer, Robert P.; Soper, Steven A.; Cheng, Yu-Wei; Barany, Francis

    2003-06-01

    New strategies for analyzing molecular signatures of disease states in real time using single pair fluorescence resonance energy transfer (spFRET) were developed to rapidly detect point mutations in unamplified genomic DNA (DNA diagnostics). The assay was carried out using allele-specific primers, which flanked the point mutation in the target gene fragment and were ligated using a thremostable ligase enzyme only when the genomic DNA carried this mutation (ligase detection reaction, LDR). We coupled LDR with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the MB probes formed upon ligation. We demonstrated the ability to rapidly discriminate single base differences in heterogeneous populations having as little as 600 copies of human genomic DNA without PCR amplification. Single base difference in the K-ras gene was discriminated in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA. Real time analyses of point mutations were also performed in PMMA microfluidic device.

  15. Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair.

    PubMed

    Akbari, Mansour; Keijzers, Guido; Maynard, Scott; Scheibye-Knudsen, Morten; Desler, Claus; Hickson, Ian D; Bohr, Vilhelm A

    2014-04-01

    Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

  16. The accessibility of thiophosphorylated groups in DNA fragments to the enzymatic activity of ligases and restriction endonuclease Bbs I.

    PubMed

    Schenk, J A; Heymann, S; Micheel, B

    1995-08-01

    The aim of this paper was to test the possibility to ligate and hydrolyse DNA sequences containing thiomodified ends and bonds. T4 DNA ligase was shown to ligate DNA fragments regardless of whether it contains phosphorylated or thiophosphorylated 5'-end. But the cleavage of an internally thiomodified phosphodiester bond was found to be totally inhibited when using the non-palindromic restrictase Bbs I. The special properties of this restriction endonuclease should allow the development of an oriented cloning strategy when combined with T4 ligase and a thiophosphorylation of DNA fragments.

  17. Naphthalimides Selectively Inhibit the Activity of Bacterial, Replicative DNA Ligases and Display Bactericidal Effects against Tubercle Bacilli.

    PubMed

    Korycka-Machala, Malgorzata; Nowosielski, Marcin; Kuron, Aneta; Rykowski, Sebastian; Olejniczak, Agnieszka; Hoffmann, Marcin; Dziadek, Jaroslaw

    2017-01-17

    The DNA ligases, enzymes that seal breaks in the backbones of DNA, are essential for all organisms, however bacterial ligases essential for DNA replication use β-nicotinamide adenine dinucleotide as their co-factor, whereas those that are essential in eukaryotes and viruses use adenosine-5'-triphosphate. This fact leads to the conclusion that NAD⁺-dependent DNA ligases in bacteria could be targeted by their co-factor specific inhibitors. The development of novel alternative medical strategies, including new drugs, are a top priority focus areas for tuberculosis research due to an increase in the number of multi-drug resistant as well as totally drug resistant tubercle bacilli strains. Here, through the use of a virtual high-throughput screen and manual inspection of the top 200 records, 23 compounds were selected for in vitro studies. The selected compounds were evaluated in respect to their Mycobacterium tuberculosis NAD⁺ DNA ligase inhibitory effect by a newly developed assay based on Genetic Analyzer 3500 Sequencer. The most effective agents (e.g., pinafide, mitonafide) inhibited the activity of M. tuberculosis NAD⁺-dependent DNA ligase A at concentrations of 50 µM. At the same time, the ATP-dependent (phage) DNA LigT₄ was unaffected by the agents at concentrations up to 2 mM. The selected compounds appeared to also be active against actively growing tubercle bacilli in concentrations as low as 15 µM.

  18. ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

    PubMed

    Petrova, T; Bezsudnova, E Y; Boyko, K M; Mardanov, A V; Polyakov, K M; Volkov, V V; Kozin, M; Ravin, N V; Shabalin, I G; Skryabin, K G; Stekhanova, T N; Kovalchuk, M V; Popov, V O

    2012-12-01

    DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

  19. Cockayne syndrome B protein regulates recruitment of the Elongin A ubiquitin ligase to sites of DNA damage.

    PubMed

    Weems, Juston C; Slaughter, Brian D; Unruh, Jay R; Boeing, Stefan; Hall, Shawn M; McLaird, Merry B; Yasukawa, Takashi; Aso, Teijiro; Svejstrup, Jesper Q; Conaway, Joan W; Conaway, Ronald C

    2017-04-21

    Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. DNA Damage Regulates UHRF1 Stability via the SCFβ-TrCP E3 Ligase

    PubMed Central

    Chen, Hao; Ma, Honghui; Inuzuka, Hiroyuki; Diao, Jianbo; Lan, Fei; Shi, Yujiang Geno; Wei, Wenyi

    2013-01-01

    UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) is a critical epigenetic player involved in the maintenance of DNA methylation patterns during DNA replication. Dysregulation of the UHRF1 level is implicated in cancer onset, metastasis, and tumor recurrence. Previous studies demonstrated that UHRF1 can be stabilized through USP7-mediated deubiquitylation, but the mechanism through which UHRF1 is ubiquitylated is still unknown. Here we show that proteasomal degradation of UHRF1 is mediated by the SCFβ-TrCP E3 ligase. Through bioinformatic and mutagenesis studies, we identified a functional DSG degron in the UHRF1 N terminus that is necessary for UHRF1 stability regulation. We further show that UHRF1 physically interacts with β-TrCP1 in a manner dependent on phosphorylation of serine 108 (S108UHRF1) within the DSG degron. Furthermore, we demonstrate that S108UHRF1 phosphorylation is catalyzed by casein kinase 1 delta (CK1δ) and is important for the recognition of UHRF1 by SCFβ-TrCP. Importantly, we demonstrate that UHRF1 degradation is accelerated in response to DNA damage, coincident with enhanced S108UHRF1 phosphorylation. Taken together, our data identify SCFβ-TrCP as a bona fide UHRF1 E3 ligase important for regulating UHRF1 steady-state levels both under normal conditions and in response to DNA damage. PMID:23297342

  1. Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase beta and ligase III/XRCC1.

    PubMed

    Prakasha Gowda, A S; Polizzi, Joanna M; Eckert, Kristin A; Spratt, Thomas E

    2010-06-15

    1-Beta-D-arabinofuranosylcytosine (cytarabine, araC) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine, dFdC), are effective cancer chemotherapeutic agents due to their ability to become incorporated into DNA and then subsequently inhibit DNA synthesis by replicative DNA polymerases. However, the impact of these 3'-modified nucleotides on the activity of specialized DNA polymerases has not been investigated. The role of polymerase beta and base excision repair may be of particular importance due to the increased oxidative stress in tumors, increased oxidative stress caused by chemotherapy treatment, and the variable amounts of polymerase beta in tumors. Here we directly investigate the incorporation of the 5'-triphosphorylated form of araC, dFdC, 2'-fluoro-2'-deoxycytidine (FdC), and cytidine into two nicked DNA substrates and the subsequent ligation. Opposite template dG, the relative k(pol)/K(d) for incorporation was dCTP > araCTP, dFdCTP > rCTP. The relative k(pol)/K(d) for FdCTP depended on sequence. The effect on k(pol)/K(d) was due largely to changes in k(pol) with no differences in the affinity of the nucleoside triphosphates to the polymerase. Ligation efficiency by T4 ligase and ligase III/XRCC1 was largely unaffected by the nucleotide analogues. Our results show that BER is capable of incorporating araC and dFdC into the genome.

  2. DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

    PubMed Central

    Bhattarai, Hitesh; Gupta, Richa

    2014-01-01

    Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. PMID:24957619

  3. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis.

    PubMed

    Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S

    2014-10-01

    Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3' phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    PubMed

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  5. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks

    PubMed Central

    Dantuma, Nico P.; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process. PMID:27148355

  6. Forkhead-Associated Domain of Yeast Xrs2, a Homolog of Human Nbs1, Promotes Nonhomologous End Joining Through Interaction With a Ligase IV Partner Protein, Lif1

    PubMed Central

    Matsuzaki, Kenichiro; Shinohara, Akira; Shinohara, Miki

    2008-01-01

    DNA double-strand breaks (DSB) are repaired through two different pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ). Yeast Xrs2, a homolog of human Nbs1, is a component of the Mre11-Rad50-Xrs2 (MRX) complex required for both HR and NHEJ. Previous studies showed that the N-terminal forkhead-associated (FHA) domain of Xrs2/Nbs1 in yeast is not involved in HR, but is likely to be in NHEJ. In this study, we showed that the FHA domain of Xrs2 plays a critical role in efficient DSB repair by NHEJ. The FHA domain of Xrs2 specifically interacts with Lif1, a component of the ligase IV complex, Dnl4-Nej1-Lif1 (DNL). Lif1, which is phosphorylated in vivo, contains two Xrs2-binding regions. Serine 383 of Lif1 plays an important role in the interaction with Xrs2 as well as in NHEJ. Interestingly, the phospho-mimetic substitutions of serine 383 enhance the NHEJ activity of Lif1. Our results suggest that the phosphorylation of Lif1 at serine 383 is recognized by the Xrs2 FHA domain, which in turn may promote recruitment of the DNL complex to DSB for NHEJ. The interaction between Xrs2 and Lif1 through the FHA domain is conserved in humans; the FHA domain Nbs1 interacts with Xrcc4, a Lif1 homolog of human. PMID:18458108

  7. A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.

    PubMed

    Tilgner, K; Neganova, I; Moreno-Gimeno, I; Al-Aama, J Y; Burks, D; Yung, S; Singhapol, C; Saretzki, G; Evans, J; Gorbunova, V; Gennery, A; Przyborski, S; Stojkovic, M; Armstrong, L; Jeggo, P; Lako, M

    2013-08-01

    DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final 'end-joining' step. Mutations in DNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affected per se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result in ex vivo expansion of stem cells with intact genomes.

  8. Combination of DNA ligase reaction and gold nanoparticle-quenched fluorescent oligonucleotides: a simple and efficient approach for fluorescent assaying of single-nucleotide polymorphisms.

    PubMed

    Wang, Hao; Li, Jishan; Wang, Yongxiang; Jin, Jiangyu; Yang, Ronghua; Wang, Kemin; Tan, Weihong

    2010-09-15

    A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination.

  9. Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

    PubMed

    Cremaschi, Paolo; Oliverio, Matteo; Leva, Valentina; Bione, Silvia; Carriero, Roberta; Mazzucco, Giulia; Palamidessi, Andrea; Scita, Giorgio; Biamonti, Giuseppe; Montecucco, Alessandra

    2015-01-01

    Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

  10. In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

    PubMed Central

    Cameron, J R; Panasenko, S M; Lehman, I R; Davis, R W

    1975-01-01

    DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells. Images PMID:1103146

  11. Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition.

    PubMed

    Palle, Komaraiah; Vaziri, Cyrus

    2011-05-15

    Camptothecin (CPT) and related chemotherapeutic drugs induce formation of DNA Topoisomerase I (Top1) covalent or cleavage complexes (Top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. The Fanconi Anemia (FA) pathway is implicated in tolerance of CPT-induced DNA damage yet the mechanism of FA pathway activation by Top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to CPT treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from CPT-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following CPT treatment. The E3 ubiquitin ligase Rad18 is necessary for CPT-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in CPT-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving PCNA monoubiquitination. In contrast, PCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of CPT-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an E3 ligase-independent mechanism, we demonstrate that Rad18 E3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after CPT treatment. Taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from PCNA ubiquitination and requirement for E3 ligase activity.

  12. NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.

    PubMed

    Srivastava, Sandeep Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2005-08-26

    DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

  13. 4-coumarate:coenzyme a ligase from loblolly pine xylem. Isolation, characterization, and complementary DNA cloning.

    PubMed Central

    Voo, K S; Whetten, R W; O'Malley, D M; Sederoff, R R

    1995-01-01

    4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) was purified from differentiating xylem of loblolly pine (Pinus taeda L.). The pine enzyme had an apparent molecular mass of 64 kD and was similar in size and kinetic properties to 4CL isolated from Norway spruce. The pine enzyme used 4-coumaric acid, caffeic acid, ferulic acid, and cinnamic acid as substrates but had no detectable activity using sinapic acid. 4CL was inhibited by naringenin and coniferin, products of phenylpropanoid metabolism. Although the lignin composition in compression wood is higher in p-hydroxyphenyl units than lignin from normal wood, there was no evidence for a different form of 4CL enzyme in differentiating xylem that was forming compression wood. cDNA clones for 4CL were obtained from a xylem expression library. The cDNA sequences matched pine xylem 4CL protein sequences and showed 60 to 66% DNA sequence identity with 4CL sequences from herbaceous angiosperms. There were two classes of cDNA obtained from pine xylem, and the genetic analysis showed that they were products of a single gene. PMID:7784527

  14. Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo.

    PubMed

    Mills, Scott D; Eakin, Ann E; Buurman, Ed T; Newman, Joseph V; Gao, Ning; Huynh, Hoan; Johnson, Kenneth D; Lahiri, Sushmita; Shapiro, Adam B; Walkup, Grant K; Yang, Wei; Stokes, Suzanne S

    2011-03-01

    DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

  15. Adenylation-Dependent Conformation and Unfolding Pathways of the NAD+-Dependent DNA Ligase from the Thermophile Thermus scotoductus

    PubMed Central

    Georlette, Daphné; Blaise, Vinciane; Bouillenne, Fabrice; Damien, Benjamin; Thorbjarnardóttir, Sigridur H.; Depiereux, Eric; Gerday, Charles; Uversky, Vladimir N.; Feller, Georges

    2004-01-01

    In the last few years, an increased attention has been focused on NAD+-dependent DNA ligases. This is mostly due to their potential use as antibiotic targets, because effective inhibition of these essential enzymes would result in the death of the bacterium. However, development of an efficient drug requires that the conformational modifications involved in the catalysis of NAD+-dependent DNA ligases are understood. From this perspective, we have investigated the conformational changes occurring in the thermophilic Thermus scotoductus NAD+-DNA ligase upon adenylation, as well as the effect of cofactor binding on protein resistance to thermal and chemical (guanidine hydrochloride) denaturation. Our results indicate that cofactor binding induces conformational rearrangement within the active site and promotes a compaction of the enzyme. These data support an induced “open-closure” process upon adenylation, leading to the formation of the catalytically active enzyme that is able to bind DNA. These conformational changes are likely to be associated with the protein function, preventing the formation of nonproductive complexes between deadenylated ligases and DNA. In addition, enzyme adenylation significantly increases resistance of the protein to thermal denaturation and GdmCl-induced unfolding, establishing a thermodynamic link between ligand binding and increased conformational stability. Finally, chemical unfolding of deadenylated and adenylated enzyme is accompanied by accumulation of at least two equilibrium intermediates, the molten globule and premolten globule states. Maximal populations of these intermediates are shifted toward higher GdmCl concentrations in the case of the adenylated ligase. These data provide further insights into the properties of partially folded intermediates. PMID:14747344

  16. Mutations of Asp540 and the domain-connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity.

    PubMed

    Tanabe, Maiko; Ishino, Sonoko; Ishino, Yoshizumi; Nishida, Hirokazu

    2014-01-21

    The structure of Pyrococcus furiosus DNA ligase (PfuLig), which architecturally resembles human DNA ligase I (hLigI), revealed that the C-terminal helix stabilizes the closed conformation through several ionic interactions between two domains (adenylylation domain (AdD) and C-terminal OB-fold domain (OBD)). This helix is oriented differently in DNA-bound hLigI, suggesting that the disruption of its interactions with AdD facilitates DNA binding. Previously, we demonstrated that the replacement of Asp540 with arginine improves the ligation activity. Here we report that the combination of the Asp540-replacement and the elimination of ionic residues in the helix, forming interactions with AdD, effectively enhanced the activity.

  17. M. tuberculosis Sliding β-Clamp Does Not Interact Directly with the NAD+ -Dependent DNA Ligase

    PubMed Central

    Kukshal, Vandna; Khanam, Taran; Chopra, Deepti; Singh, Nidhi; Sanyal, Sabyasachi; Ramachandran, Ravishankar

    2012-01-01

    The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C2221 with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent Kd of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD+ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria. PMID:22545130

  18. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

    PubMed Central

    Mallik, Sarita; Popodi, Ellen M.; Hanson, Andrew J.

    2015-01-01

    ABSTRACT Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. IMPORTANCE DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings

  19. Pharmacophore-based screening and identification of novel human ligase I inhibitors with potential anticancer activity.

    PubMed

    Krishna, Shagun; Singh, Deependra Kumar; Meena, Sanjeev; Datta, Dipak; Siddiqi, Mohammad Imran; Banerjee, Dibyendu

    2014-03-24

    Human DNA ligases are enzymes that are indispensable for DNA replication and repair processes. Among the three human ligases, ligase I is attributed to the ligation of thousands of Okazaki fragments that are formed during lagging strand synthesis during DNA replication. Blocking ligation therefore can lead to the accumulation of thousands of single strands and subsequently double strand breaks in the DNA, which is lethal for the cells. The reports of the high expression level of ligase I protein in several cancer cells (versus the low ligase expression level and the low rate of division of most normal cells in the adult body) support the belief that ligase I inhibitors can target cancer cells specifically with minimum side effects to normal cells. Recent publications showing exciting data for a ligase IV inhibitor exhibiting antitumor activity in mouse models also strengthens the argument for ligases as valid antitumor targets. Keeping this in view, we performed a pharmacophore-based screening for potential ligase inhibitors in the Maybridge small molecule library and procured some of the top-ranking compounds for enzyme-based and cell-based in vitro screening. We report here the identification of novel ligase I inhibitors with potential anticancer activity against a colon cancer cell line.

  20. Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

    PubMed

    Ren, Z J; Baumann, R G; Black, L W

    1997-08-22

    A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.

  1. DNA gyrase, topoisomerase IV, and the 4-quinolones.

    PubMed Central

    Drlica, K; Zhao, X

    1997-01-01

    For many years, DNA gyrase was thought to be responsible both for unlinking replicated daughter chromosomes and for controlling negative superhelical tension in bacterial DNA. However, in 1990 a homolog of gyrase, topoisomerase IV, that had a potent decatenating activity was discovered. It is now clear that topoisomerase IV, rather than gyrase, is responsible for decatenation of interlinked chromosomes. Moreover, topoisomerase IV is a target of the 4-quinolones, antibacterial agents that had previously been thought to target only gyrase. The key event in quinolone action is reversible trapping of gyrase-DNA and topoisomerase IV-DNA complexes. Complex formation with gyrase is followed by a rapid, reversible inhibition of DNA synthesis, cessation of growth, and induction of the SOS response. At higher drug concentrations, cell death occurs as double-strand DNA breaks are released from trapped gyrase and/or topoisomerase IV complexes. Repair of quinolone-induced DNA damage occurs largely via recombination pathways. In many gram-negative bacteria, resistance to moderate levels of quinolone arises from mutation of the gyrase A protein and resistance to high levels of quinolone arises from mutation of a second gyrase and/or topoisomerase IV site. For some gram-positive bacteria, the situation is reversed: primary resistance occurs through changes in topoisomerase IV while gyrase changes give additional resistance. Gyrase is also trapped on DNA by lethal gene products of certain large, low-copy-number plasmids. Thus, quinolone-topoisomerase biology is providing a model for understanding aspects of host-parasite interactions and providing ways to investigate manipulation of the bacterial chromosome by topoisomerases. PMID:9293187

  2. Destabilization of CDC6 upon DNA damage is dependent on neddylation but independent of Cullin E3 ligases.

    PubMed

    Tan, Chia Yee; Hagen, Thilo

    2013-07-01

    CDC6 is an important component of the pre-replication complex and plays an essential role in the regulation of DNA replication in eukaryotic cells. Deregulation of CDC6 protein levels results in rereplication and genomic instability. CDC6 expression is tightly regulated during the cell cycle. One major mechanism of cell cycle dependent regulation of CDC6 is APC(Cdh1) mediated protein ubiquitination and degradation during G1 phase. In addition to APC(Cdh1) dependent degradation, alternative, Cullin RING E3 ubiquitin ligase dependent degradation pathways have been characterized in yeast. Here we studied whether Cullin RING E3 ligases also play a role in the turnover of CDC6 protein in mammalian cells. To this end, we used the Nedd8 E1 inhibitor MLN4924, which blocks the activity of all Cullin E3 ligases. We observed that treatment with MLN4924 increased CDC6 protein expression. However, this effect was due to a delay in cell cycle progression from G1 to S phase, resulting in accumulation of cells with high CDC6 protein levels. Therefore, our results indicate that Cullin E3 ligases are not involved in the basal turnover of CDC6 in mammalian cells. Interestingly, we also found that the DNA cross-linker mitomycin C induces marked CDC6 protein degradation. Mitomycin C induced CDC6 degradation is not mediated by APC(Cdh1), Cullin or HUWE1 E3 ubiquitin ligases. Notably, mitomycin C mediated CDC6 degradation requires the neddylation pathway. Our results provide evidence for a novel, cullin independent mechanism of CDC6 posttranslational regulation upon DNA damage that involves the neddylation pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. An exonic splicing silencer in the testes-specific DNA ligase III β exon

    PubMed Central

    Chew, Shern L.; Baginsky, Lysa; Eperon, Ian C.

    2000-01-01

    Alternative pre-mRNA splicing of two terminal exons (α and β) regulates the expression of the human DNA ligase III gene. In most tissues, the α exon is expressed. In testes and during spermatogenesis, the β exon is used instead. The α exon encodes the interaction domain with a scaffold DNA repair protein, XRCC1, while the β exon-encoded C-terminal does not. Sequence elements regulating the alternative splicing pattern were mapped by in vitro splicing assays in HeLa nuclear extracts. Deletion of a region beginning in the β exon and extending into the downstream intron derepressed splicing to the β exon. Two silencing elements were found within this 101 nt region: a 16 nt exonic splicing silencer immediately upstream of the β exon polyadenylation signal and a 45 nt intronic splicing silencer. The exonic splicing silencer inhibited splicing, even when the polyadenylation signal was deleted or replaced by a 5′ splice site. This element also enhanced polyadenylation under conditions unfavourable to splicing. The splicing silencer partially inhibited assembly of spliceosomal complexes and functioned in an adenoviral pre-mRNA context. Silencing of splicing by the element was associated with cross-linking of a 37 kDa protein to the RNA substrate. The element exerts opposite functions in splicing and polyadenylation. PMID:10606636

  4. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  5. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions.

    PubMed

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-09

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2-5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo.

  6. Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    PubMed Central

    Konomura, Naoto; Arai, Naoto; Shinohara, Takeshi; Kobayashi, Jun; Iwasaki, Wakana; Ikawa, Shukuko; Kusano, Kohji; Shibata, Takehiko

    2017-01-01

    RecA-family recombinase-catalyzed ATP-dependent homologous joint formation is critical for homologous recombination, in which RecA or Rad51 binds first to single-stranded (ss)DNA and then interacts with double-stranded (ds)DNA. However, when RecA or Rad51 interacts with dsDNA before binding to ssDNA, the homologous joint-forming activity of RecA or Rad51 is quickly suppressed. We found that under these and adenosine diphosphate (ADP)-generating suppressive conditions for the recombinase activity, RecA or Rad51 at similar optimal concentrations enhances the DNA ligase-catalyzed dsDNA end-joining (DNA ligation) about 30- to 40-fold. The DNA ligation enhancement by RecA or Rad51 transforms most of the substrate DNA into multimers within 2–5 min, and for this enhancement, ADP is the common and best cofactor. Adenosine triphosphate (ATP) is effective for RecA, but not for Rad51. Rad51/RecA-enhanced DNA ligation depends on dsDNA-binding, as shown by a mutant, and is independent of physical interactions with the DNA ligase. These observations demonstrate the common and unique activities of RecA and Rad51 to juxtapose dsDNA-ends in preparation for covalent joining by a DNA ligase. This new in vitro function of Rad51 provides a simple explanation for our genetic observation that Rad51 plays a role in the fidelity of the end-joining of a reporter plasmid DNA, by yeast canonical non-homologous end-joining (NHEJ) in vivo. PMID:27794044

  7. Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

    DOE PAGES

    Aceytuno, R.  Daniel; Piett, Cortt G.; Havali-Shahriari, Zahra; ...

    2017-04-27

    Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5'-phosphate/3'-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation andmore » that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexiblemultistate complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. Amutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.« less

  8. Efficient ligase 3-dependent microhomology-mediated end joining repair of DNA double-strand breaks in zebrafish embryos.

    PubMed

    He, Mu-Dan; Zhang, Feng-Hua; Wang, Hua-Lin; Wang, Hou-Peng; Zhu, Zuo-Yan; Sun, Yong-Hua

    2015-10-01

    DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro-designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Mechanistic Assessment of DNA Ligase as an Antibacterial Target in Staphylococcus aureus

    PubMed Central

    Podos, Steven D.; Thanassi, Jane A.

    2012-01-01

    We report the use of a known pyridochromanone inhibitor with antibacterial activity to assess the validity of NAD+-dependent DNA ligase (LigA) as an antibacterial target in Staphylococcus aureus. Potent inhibition of purified LigA was demonstrated in a DNA ligation assay (inhibition constant [Ki] = 4.0 nM) and in a DNA-independent enzyme adenylation assay using full-length LigA (50% inhibitory concentration [IC50] = 28 nM) or its isolated adenylation domain (IC50 = 36 nM). Antistaphylococcal activity was confirmed against methicillin-susceptible and -resistant S. aureus (MSSA and MRSA) strains (MIC = 1.0 μg/ml). Analysis of spontaneous resistance potential revealed a high frequency of emergence (4 × 10−7) of high-level resistant mutants (MIC > 64) with associated ligA lesions. There were no observable effects on growth rate in these mutants. Of 22 sequenced clones, 3 encoded point substitutions within the catalytic adenylation domain and 19 in the downstream oligonucleotide-binding (OB) fold and helix-hairpin-helix (HhH) domains. In vitro characterization of the enzymatic properties of four selected mutants revealed distinct signatures underlying their resistance to inhibition. The infrequent adenylation domain mutations altered the kinetics of adenylation and probably elicited resistance directly. In contrast, the highly represented OB fold domain mutations demonstrated a generalized resistance mechanism in which covalent LigA activation proceeds normally and yet the parameters of downstream ligation steps are altered. A resulting decrease in substrate Km and a consequent increase in substrate occupancy render LigA resistant to competitive inhibition. We conclude that the observed tolerance of staphylococcal cells to such hypomorphic mutations probably invalidates LigA as a viable target for antistaphylococcal chemotherapy. PMID:22585221

  10. Molecular Characterization of NAD+-Dependent DNA Ligase from Wolbachia Endosymbiont of Lymphatic Filarial Parasite Brugia malayi

    PubMed Central

    Shrivastava, Nidhi; Nag, Jeetendra Kumar; Misra-Bhattacharya, Shailja

    2012-01-01

    The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD+-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD+-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD+-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies. PMID:22815933

  11. Development of a PCR/ligase detection reaction/nanogold-based universal array approach for the detection of low-abundant DNA point mutations.

    PubMed

    Yi, Ping; Lu, Weiping; Guo, Jianxin; Liu, Qiang; Chen, Zhuqin; Han, Jian; Li, Li

    2011-12-01

    The aim of this study was to investigate the feasibility of combining PCR and ligase detection reaction (LDR) with a novel nano-gold-based universal array for the detection of low abundance point mutations from fetal DNA in maternal plasma samples. The sequence with the target point mutation was first amplified by PCR and then used as a template for LDR in which the upstream specific primer contains a tag sequence at the 5'-end. After hybridization to the probes of a universal array containing anti-tag sequences, the ligated products were bound to streptavidin-labeled nano-gold particles and the hybridization signals were amplified by silver staining. The PCR/LDR/universal array was first tested for sensitivity with nano-gold-based detection, and then this system was applied to detect the low abundance specific mutation IVS2 654(C→T) of the β-globin gene in a model using maternal plasma samples. The nano-gold-based method unambiguously identified a single mutation at a sensitivity of 1:1000. This approach was applied to detect the paternally inherited IVS2 654(C→T) mutation from thirty maternal plasma samples. The results were consistent with those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. The PCR/LDR/nano-gold-based universal array is able to detect low-abundance point mutations with high sensitivity.

  12. Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

    PubMed

    Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

    2017-08-17

    DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Fluoroquinolones inhibit preferentially Streptococcus pneumoniae DNA topoisomerase IV than DNA gyrase native proteins.

    PubMed

    Fernandez-Moreira, E; Balas, D; Gonzalez, I; de la Campa, A G

    2000-01-01

    The genes encoding the subunits of DNA topoisomerase IV (parC and parE) and DNA gyrase (gyrA and gyrB) of Streptococcus pneumoniae were cloned and overproduced in Escherichia coli by using the T7promoter-T7 RNA polymerase system. The four subunits were separately purified to near homogeneity by column chromatography. Protein purification was achieved by DEAE-sepharose, heparin-agarose, and hydroxylapatite chromatography. DNA topoisomerase IV was reconstituted when ParC and ParE were combined at a 3.8-fold excess of ParE. The reconstituted topoisomerase IV showed to generate efficient ATP-dependent DNA decatenation activity. The DNA gyrase ATP-dependent supercoiling activity was reconstituted by mixing equimolar amounts of the two gyrase subunits. The inhibitory effects of four representative fluoroquinolones on the DNA decatenation activity of topoisomerase IV and DNA supercoiling of gyrase have been examined and compared. All four compounds were more active in inhibiting topoisomerase IV than gyrase. Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and DNA gyrase supercoiling. The classification of the four fluoroquinolones, considering their inhibitory activities in decatenation, supercoiling and growth was the following: clinafloxacin > trovafloxacin > sparfloxacin > ciprofloxacin. These results suggest these drugs primarily target topoisomerase IV of S. pneumoniae, and gyrase secondarily, in agreement with genetic data.

  14. A new nuclear DNA marker from ubiquitin ligase gene region for genetic diversity detection of walnut germplasm resources.

    PubMed

    Suo, Zhili; Chen, Lingna; Pei, Dong; Jin, Xiaobai; Zhang, Huijin

    2015-03-01

    Development of more sensitive nuclear DNA markers for identification of species, particularly closely allied taxa has been a challenging task that has attracted interest from scientists in fields of biotechnological development and genetic diversity detection. In this study, the sequence of the ubiquitin ligase gene (UBE3) region of nuclear DNA was tested for applicability and efficacy in revealing genetic diversity of walnut resources, with an emphasis on inter- and intra-specific levels. Analysis on genetic relationship among the taxa was conducted with the neighbor-joining (NJ) method. The number of variable bases in the UBE3 region was 20 sites. All nine taxa (species/variety/cultivars) were distinguished using the UBE3 sequence. In addition, each taxon was characterized molecularly with a unique nucleotide molecular formula using ten variable base sites derived from the nuclear DNA UBE3 gene sequence. This study presents a good complementary methodology for developing new DNA markers for identification of genus Juglans.

  15. A High-Fidelity Codon Set for the T4 DNA Ligase-Catalyzed Polymerization of Modified Oligonucleotides.

    PubMed

    Lei, Yi; Kong, Dehui; Hili, Ryan

    2015-12-14

    In vitro selection of nucleic acid polymers can readily deliver highly specific receptors and catalysts for a variety of applications; however, it is suspected that the functional group deficit of nucleic acids has limited their potential with respect to proteinogenic polymers. This has stimulated research toward expanding their chemical diversity to bridge the functional gap between nucleic acids and proteins to develop a superior biopolymer. In this study, we investigate the effect of codon library size and composition on the sequence specificity of T4 DNA ligase in the DNA-templated polymerization of both unmodified and modified oligonucleotides. Using high-throughput DNA sequencing of duplex pairs, we have uncovered a 256-membered codon set that yields sequence-defined modified ssDNA polymers in high yield and with high fidelity.

  16. Atomic Structure and Nonhomologous End-Joining Function of the Polymerase Component of Bacterial DNA Ligase D

    SciTech Connect

    Zhu,H.; Nandakumar, J.; Aniukwu, J.; Wang, L.; Glickman, M.; Lima, C.; Shuman, S.

    2006-01-01

    DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous end-joining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5- Angstroms crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP-dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end double-strand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair.

  17. The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase.

    PubMed

    Pawlowska, Roza; Korczynski, Dariusz; Nawrot, Barbara; Stec, Wojciech J; Chworos, Arkadiusz

    2016-08-01

    T4 DNA ligase is one of the most commonly used enzymes for in vitro molecular research and a useful model for testing the ligation mechanism of ATP-dependent DNA ligation. To better understand the influence of phosphate group modifications in the ligation process, a series of ATP analogs were tested as cofactors. P-diastereomers of newly developed β,γ-hypo-ATPαS (thio) and β,γ-hypo-ATP (oxo) were synthesized and their activity was compared to ATPαS and their natural precursors. The evaluation of presented ATP analogs revealed the importance of the α-phosphate stereogenic center in ATPαS for the T4 DNA ligase activity and sheds new light on the interaction between ATP-dependent DNA ligases and cofactors.

  18. Pirh2: an E3 ligase with central roles in the regulation of cell cycle, DNA damage response, and differentiation.

    PubMed

    Halaby, Marie-jo; Hakem, Razqallah; Hakem, Anne

    2013-09-01

    Ubiquitylation is currently recognized as a major posttranslational modification that regulates diverse cellular processes. Pirh2 is a ubiquitin E3 ligase that regulates the turnover and functionality of several proteins involved in cell proliferation and differentiation, cell cycle checkpoints, and cell death. Here we review the role of Pirh2 as a regulator of the DNA damage response through the ubiquitylation of p53, Chk2, p73, and PolH. By ubiquitylating these proteins, Pirh2 regulates cell cycle checkpoints and cell death in response to DNA double-strand breaks or the formation of bulky DNA lesions. We also discuss how Pirh2 affects cell proliferation and differentiation in unstressed conditions through ubiquitylation and degradation of c-Myc, p63, and p27(kip1). Finally, we link these different functions of Pirh2 to its role as a tumor suppressor in mice and as a prognosis marker in various human cancer subtypes.

  19. The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.

    PubMed

    Lei, Chao; Li, Shi-Yuan; Liu, Jia-Kun; Zheng, Xuan; Zhao, Guo-Ping; Wang, Jin

    2017-01-23

    As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.

  20. SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage

    PubMed Central

    Yin, Yili; Seifert, Anne; Chua, Joy Shijia; Maure, Jean-François; Golebiowski, Filip; Hay, Ronald T.

    2012-01-01

    Here we demonstrate that RNF4, a highly conserved small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, plays a critical role in the response of mammalian cells to DNA damage. Human cells in which RNF4 expression was ablated by siRNA or chicken DT40 cells with a homozygous deletion of the RNF4 gene displayed increased sensitivity to DNA-damaging agents. Recruitment of RNF4 to double-strand breaks required its RING and SUMO interaction motif (SIM) domains and DNA damage factors such as NBS1, mediator of DNA damage checkpoint 1 (MDC1), RNF8, 53BP1, and BRCA1. In the absence of RNF4, these factors were still recruited to sites of DNA damage, but 53BP1, RNF8, and RNF168 displayed delayed clearance from such foci. SILAC-based proteomics of SUMO substrates revealed that MDC1 was SUMO-modified in response to ionizing radiation. As a consequence of SUMO modification, MDC1 recruited RNF4, which mediated ubiquitylation at the DNA damage site. Failure to recruit RNF4 resulted in defective loading of replication protein A (RPA) and Rad51 onto ssDNA. This appeared to be a consequence of reduced recruitment of the CtIP nuclease, resulting in inefficient end resection. Thus, RNF4 is a novel DNA damage-responsive protein that plays a role in homologous recombination and integrates SUMO modification and ubiquitin signaling in the cellular response to genotoxic stress. PMID:22661230

  1. Specific DNA recognition mediated by a type IV pilin

    PubMed Central

    Cehovin, Ana; Simpson, Peter J.; McDowell, Melanie A.; Brown, Daniel R.; Noschese, Rossella; Pallett, Mitchell; Brady, Jacob; Baldwin, Geoffrey S.; Lea, Susan M.; Matthews, Stephen J.; Pelicic, Vladimir

    2013-01-01

    Natural transformation is a dominant force in bacterial evolution by promoting horizontal gene transfer. This process may have devastating consequences, such as the spread of antibiotic resistance or the emergence of highly virulent clones. However, uptake and recombination of foreign DNA are most often deleterious to competent species. Therefore, model naturally transformable Gram-negative bacteria, including the human pathogen Neisseria meningitidis, have evolved means to preferentially take up homotypic DNA containing short and genus-specific sequence motifs. Despite decades of intense investigations, the DNA uptake sequence receptor in Neisseria species has remained elusive. We show here, using a multidisciplinary approach combining biochemistry, molecular genetics, and structural biology, that meningococcal type IV pili bind DNA through the minor pilin ComP via an electropositive stripe that is predicted to be exposed on the filaments surface and that ComP displays an exquisite binding preference for DNA uptake sequence. Our findings illuminate the earliest step in natural transformation, reveal an unconventional mechanism for DNA binding, and suggest that selective DNA uptake is more widespread than previously thought. PMID:23386723

  2. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair

    PubMed Central

    Galanty, Yaron; Belotserkovskaya, Rimma; Coates, Julia; Jackson, Stephen P.

    2012-01-01

    Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems. PMID:22661229

  3. Kinetics of T3-DNA Ligase-Catalyzed Phosphodiester Bond Formation Measured Using the α-Hemolysin Nanopore.

    PubMed

    Tan, Cherie S; Riedl, Jan; Fleming, Aaron M; Burrows, Cynthia J; White, Henry S

    2016-12-27

    The latch region of the wild-type α-hemolysin (α-HL) protein channel can be used to distinguish single base modifications in double-stranded DNA (dsDNA) via ion channel measurements upon electrophoretic capture of dsDNA in the vestibule of α-HL. Herein, we investigated the use of the latch region to detect a nick in the phosphodiester DNA backbone. The presence of a nick in the phosphodiester backbone of one strand of the duplex results in a significant increase in both the blockade current and noise level relative to the intact duplex. Differentiation between the nicked and intact duplexes based on blockade current or noise, with near baseline resolution, allows real-time monitoring of the rate of T3-DNA ligase-catalyzed phosphodiester bond formation. Under low ionic strength conditions containing divalent cations and a molecular crowding agent (75 mg mL(-1) PEG), the rate of enzyme-catalyzed reaction in the bulk solution was continuously monitored by electrophoretically capturing reaction substrate or product dsDNA in the α-HL protein channel vestibule. Enzyme kinetic results obtained from the nanopore experiments match those from gel electrophoresis under the same reaction conditions, indicating the α-HL nanopore measurement provides a viable approach for monitoring enzymatic DNA repair activity.

  4. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2001-07-01

    resting breast tissues from 10 different patients express both components of DNA-PK, DNAPKcs and Ku. These tissues also expressed XRCC4, DNA Ligase IV...DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as three other DNA repair components NBS1, MRE11, and PCNA. In contrast to the original report, 90% of the epithelial cells in normal

  5. Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification.

    PubMed

    Jiang, Hong-Xin; Kong, De-Ming; Shen, Han-Xi

    2014-05-15

    As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

  6. Rad7 E3 Ubiquitin Ligase Attenuates Polyubiquitylation of Rpn10 and Dsk2 Following DNA Damage in Saccharomyces cerevisiae

    PubMed Central

    Benoun, Joseph M.; Lalimar-Cortez, Danielle; Valencia, Analila; Granda, Adriana; Moore, Destaye M.; Kelson, Eric P.

    2016-01-01

    During Nucleotide Excision Repair (NER) in the yeast S. cerevisiae, ubiquitylation of Rad4 is carried out by the E3 ubiquitin ligase that includes Rad7-Elc1-Cul3 and is critical to optimal NER. Rad7 E3 activity targets Rad4 for degradation by the proteaseome but, in principle, could also trigger other DNA damage responses. We observed increased nuclear ubiquitin foci (Ub-RFP) formation in S. cerevisiae containing a Rad7 E3 ligase mutant (rad7SOCS) in response to DNA damage by benzo[a]pyrenediolepoxide (BPDE) in dividing cells. Immunoblots reveal that ubiquitin conjugates of Rpn10 and Dsk2 accumulate in greater abundance in rad7SOCS compared to RAD7 in dividing cells in response to BPDE which makes Rpn10 and Dsk2 candidates for being the ubiquitylated species observed in our microscopy experiments. Microscopy analysis with strains containing Dsk2-GFP shows that Dsk2-GFP and Dsk2-GFP/Ub-RFP colocalized in nuclear foci form to an increased extent in a rad7SOCS mutant background in dividing cells than in a RAD7 wild-type strain. Further, Dsk2-GFP in the rad7SOCS strain formed more foci at the plasma membrane following BPDE treatment in dividing cells relative to strains containing RAD7 or a rad7Δ deletion mutant. In response to a different agent, UV irradiation, levels of ubiquitylated proteins were increased in rad7SOCS relative to RAD7, and the proteasomal deubiquitylase subunit, Rpn11 was even monoubiquitylated in the absence of damaging agents. Together these data show that Rad7 E3 activity attenuates ubiquitylation of proteins regulating the shuttling of polyubiquitylated proteins to the proteasome (Dsk2 and Rpn10) and removal of ubiquitin chains just prior to degradation (Rpn11). Since Rad7 E3 ligase activity has been shown to increase ubiquitylation of its target proteins, yet our results show increased ubiquitylation in the absence of Rad7 E3, we suggest that Rad7 E3 action regulates ubiquitin ligase and deubiquitylase (DUB) activities that act on Rpn10, Dsk2

  7. Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    PubMed Central

    Fu, Changlin; Donovan, William P.; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H.

    2014-01-01

    Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%. PMID:25551825

  8. Regulation of DNA topoisomerase IIalpha stability by the ECV ubiquitin ligase complex.

    PubMed

    Yun, Jisoo; Kim, Yong-Il; Tomida, Akihiro; Choi, Cheol-Hee

    2009-11-06

    In this study, we attempted to elucidate the E3 ubiquitin ligase for topo IIalpha. When cullins and VHL were ectopically expressed in HT1080 and HEK293T cells, topo IIalpha was degraded most prominently in cullin 2- and VHL-expressing cells. Cullin 2 and the beta domain (aa 114-123) of VHL, a subunit of the ECV (Elongin B/C-cullin 2-VHL protein) complex, specifically interact with the ATPase domain of topo IIalpha. We identified that topo IIalpha associated with endogenous Elongin C. In HT1080 cells co-transfected with deletion mutants of topo IIalpha GRDD (glucose-regulated destruction domain) and VHL, topo IIalpha was degraded by VHL expression. These results demonstrate that ECV acts as E3 ubiquitin ligase targeting GRDD-independent topo IIalpha to the ubiquitin-proteasome pathway.

  9. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    PubMed

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  10. Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    PubMed Central

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J.H. David; Shao, Weilan

    2013-01-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5′ end of the PCR primer and the extended newly synthesized DNA 3′ end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by ‘selection marker swapping’ upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution. PMID:23633530

  11. Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus.

    PubMed

    Georlette, Daphné; Blaise, Vinciane; Dohmen, Christophe; Bouillenne, Fabrice; Damien, Benjamin; Depiereux, Eric; Gerday, Charles; Uversky, Vladimir N; Feller, Georges

    2003-12-12

    DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.

  12. DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

    PubMed Central

    Abdou, Ismail; Poirier, Guy G.; Hendzel, Michael J.; Weinfeld, Michael

    2015-01-01

    In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme's SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3. PMID:25539916

  13. RNAi-Based Suppressor Screens Reveal Genetic Interactions Between the CRL2LRR-1 E3-Ligase and the DNA Replication Machinery in Caenorhabditis elegans

    PubMed Central

    Ossareh-Nazari, Batool; Katsiarimpa, Anthi; Merlet, Jorge; Pintard, Lionel

    2016-01-01

    Cullin-RING E3-Ligases (CRLs), the largest family of E3 ubiquitin-Ligases, regulate diverse cellular processes by promoting ubiquitination of target proteins. The evolutionarily conserved Leucine Rich Repeat protein 1 (LRR-1) is a substrate-recognition subunit of a CRL2LRR-1 E3-ligase. Here we provide genetic evidence supporting a role of this E3-enzyme in the maintenance of DNA replication integrity in Caenorhabditis elegans. Through RNAi-based suppressor screens of lrr-1(0) and cul-2(or209ts) mutants, we identified two genes encoding components of the GINS complex, which is part of the Cdc45-MCM-GINS (CMG) replicative helicase, as well as CDC-7 and MUS-101, which drives the assembly of the CMG helicase during DNA replication. In addition, we identified the core components of the ATR/ATL-1 DNA replication checkpoint pathway (MUS-101, ATL-1, CLSP-1, CHK-1). These results suggest that the CRL2LRR-1 E3-ligase acts to modify or degrade factor(s) that would otherwise misregulate the replisome, eventually leading to the activation of the DNA replication checkpoint. PMID:27543292

  14. Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    PubMed Central

    de Ory, Ana; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; de Vega, Miguel

    2016-01-01

    Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs. PMID:26826709

  15. Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts.

    PubMed

    Sillero, Maria A Günther; de Diego, Anabel; Tavares, Janeth E F; Silva, Joana A D Catanho da; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2009-08-15

    Compounds of the mevalonate pathway containing a terminal di- or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6+/-1.4 mU/mg or 100%; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V(max), K(m), K(cat) and K(cat)/K(m) values were also determined. The K(cat)/K(m) values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl- or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells.

  16. Trip12 is an E3 ubiquitin ligase for USP7/HAUSP involved in the DNA damage response.

    PubMed

    Liu, Xiaoliang; Yang, Xiangcai; Li, Yongxin; Zhao, Shuhua; Li, Chaocui; Ma, Pengcheng; Mao, Bingyu

    2016-12-01

    The deubiquitinating enzyme, USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), is a key regulator of the tumor suppressor p53 and plays a major role in regulating genome stability. Here, we report that the protein stability of USP7 is regulated by the ubiquitin-proteasome pathway. We identified the thyroid hormone receptor interactor 12 (Trip12) as a ubiquitin E3 ligase for USP7. We also found that Trip12 affects USP7-mediated stabilization of p53 and the checkpoint proteins 53BP1 and Chk1. Knockdown of Trip12 leads to an increased cell population in G1 phase, mimicking USP7 overexpression. In contrast, Trip12 overexpression increased the number of cells in intra-S-phase, phenocopying the USP7 knockdown phenotype. Therefore, our data reveal an important modulatory role for Trip12 in the USP7-dependent DNA damage response. © 2016 Federation of European Biochemical Societies.

  17. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection

    PubMed Central

    Ferretti, Lorenza P.; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I.; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A.

    2016-01-01

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein–protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity. PMID:27561354

  18. Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.

    PubMed

    Ferretti, Lorenza P; Himmels, Sarah-Felicitas; Trenner, Anika; Walker, Christina; von Aesch, Christine; Eggenschwiler, Aline; Murina, Olga; Enchev, Radoslav I; Peter, Matthias; Freire, Raimundo; Porro, Antonio; Sartori, Alessandro A

    2016-08-26

    Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.

  19. Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase 4

    PubMed Central

    Boboila, Cristian; Yan, Catherine; Wesemann, Duane R.; Jankovic, Mila; Wang, Jing H.; Manis, John; Nussenzweig, Andre; Nussenzweig, Michel

    2010-01-01

    The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR. PMID:20142431

  20. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D

    PubMed Central

    Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3’-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotide and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologs of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both, in an M. smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5’-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ. PMID:23198659

  1. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D.

    PubMed

    Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S

    2012-12-21

    Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ.

  2. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV.

    PubMed

    Tashjian, Tommy F; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M; Godoy, Veronica G

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  3. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    PubMed Central

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability. PMID:28298904

  4. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  5. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage.

    PubMed

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M; Bunting, Samuel F; Inuzuka, Hiroyuki; Zheng, X F Steven; Shen, Zhiyuan; Sabaawy, Hatem E; Liu, LianXin; Pine, Sharon R

    2016-10-14

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance.

  6. Efficient single-strand cleavage of DNA mediated by a MnIIIMnIV-based artificial nuclease.

    PubMed

    Qian, Jing; Yu, Shasha; Wang, Wenjun; Wang, Liping; Tian, Jinlei; Yan, Shiping

    2014-02-14

    A water-soluble Mn(IV) 1,4,7-triazacyclononane complex, [Mn(IV)2L2(μ-O)2](ClO4)2·2H2O (1), was prepared to serve as a nuclease mimic (L = 1,4,7-triazacyclononane-N-acetate). Complex 1 was readily synthesized from the highly water soluble ligand (L), with Mn(III) salt, [Mn3O(MeCO2)7]·3H2O in basic condition, and characterized by X-ray, IR, electronic spectroscopy, cyclic voltammetry and magnetic susceptibility as well as ESI-MS. The bond valence sum (BVS) analysis and magnetic data suggest that 1 is a Mn(IV)-μ-O2-Mn(IV) species. The electrospray mass spectrum and electronic spectrum of 1 in aqueous solution indicates that dinuclear Mn complex [Mn(III)Mn(IV)L2(μ-O)2](+) (2) is the active species. A predominantly hydrolytic cleavage mechanism was confirmed through experiments performed in the presence of various radical scavengers, T4 ligase and under anaerobic conditions. The kinetic aspects of DNA cleavage under pseudo- or true-Michaelis-Menten conditions were also detailed, kinetic parameters (kcat, KM, Vmax) were calculated to be 6.27 h(-1), 7.35 × 10(-5) M, 4.6 × 10(-4) M h(-1); 0.683 h(-1), 1.93 × 10(-5) M, 1.32 × 10(-5) M h(-1) for 2, respectively.

  7. Analysis of the distribution and evolution of the ATP-dependent DNA ligases of bacteria delineates a distinct phylogenetic group 'Lig E'.

    PubMed

    Williamson, Adele; Hjerde, Erik; Kahlke, Tim

    2016-01-01

    Prior to the discovery of a minimal ATP-dependent DNA ligase in Haemophilus influenzae, bacteria were thought to only possess a NAD-dependent ligase, which was involved in sealing of Okazaki fragments. We now know that a diverse range of bacterial species possess up to six of these accessory bacterial ATP-dependent DNA ligases (b-ADLs), which vary in size and enzymatic domain associations. Here we compare the domain structure of different types of b-ADLs and investigate their distribution among the bacterial domain to describe possible evolutionary trajectories that gave rise to the sequence and structural diversity of these enzymes. Previous biochemical and genetic analyses have delineated three main classes of these enzymes: Lig B, Lig C and Lig D, which appear to have descended from a common ancestor within the bacterial domain. In the present study, we delineate a fourth group of b-ADLs, Lig E, which possesses a number of unique features at the primary and tertiary structural levels. The biochemical characteristics, domain structure and inferred extracellular location sets this group apart from the other b-ADLs. The results presented here indicate that the Lig E type ligases were horizontally transferred into bacteria in a separate event from other b-ADLs possibly from a bacteriophage. © 2015 John Wiley & Sons Ltd.

  8. Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

    PubMed Central

    Chauleau, Mathieu; Shuman, Stewart

    2016-01-01

    Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO4 nicks in duplex DNA via reaction of LigA with NAD+ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO4 to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO4 nicks, kstep2 was fast (6.8–27 s−1) and similar to kstep3 (8.3–42 s−1). Absent ammonium, kstep2 and kstep3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in kstep2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA's potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions. PMID:26857547

  9. Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

    PubMed

    Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

    2008-08-22

    NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

  10. Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4

    PubMed Central

    Liddiard, Kate; Ruis, Brian; Takasugi, Taylor; Harvey, Adam; Ashelford, Kevin E.; Hendrickson, Eric A.; Baird, Duncan M.

    2016-01-01

    Telomeres shorten with each cell division and can ultimately become substrates for nonhomologous end-joining repair, leading to large-scale genomic rearrangements of the kind frequently observed in human cancers. We have characterized more than 1400 telomere fusion events at the single-molecule level, using a combination of high-throughput sequence analysis together with experimentally induced telomeric double-stranded DNA breaks. We show that a single chromosomal dysfunctional telomere can fuse with diverse nontelomeric genomic loci, even in the presence of an otherwise stable genome, and that fusion predominates in coding regions. Fusion frequency was markedly increased in the absence of TP53 checkpoint control and significantly modulated by the cellular capacity for classical, versus alternative, nonhomologous end joining (NHEJ). We observed a striking reduction in inter-chromosomal fusion events in cells lacking DNA ligase 4, in contrast to a remarkably consistent profile of intra-chromosomal fusion in the context of multiple genetic knockouts, including DNA ligase 3 and 4 double-knockouts. We reveal distinct mutational signatures associated with classical NHEJ-mediated inter-chromosomal, as opposed to alternative NHEJ-mediated intra-chromosomal, telomere fusions and evidence for an unanticipated sufficiency of DNA ligase 1 for these intra-chromosomal events. Our findings have implications for mechanisms driving cancer genome evolution. PMID:26941250

  11. The DNA binding domain of human DNA ligase I interacts with both nicked DNA and the DNA sliding clamps, PCNA and hRad9-hRad1-hHus1

    PubMed Central

    Song, Wei; Pascal, John M.; Ellenberger, Tom; Tomkinson, Alan E.

    2009-01-01

    The participation of the DNA ligase (hLigI) encoded by the human LIG1 gene in DNA replication and repair is mediated by an interaction with proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp. Interestingly, the catalytic fragment of hLigI encircles a DNA nick forming a ring that is similar in size and shape to the PCNA ring. Here we show that the DNA binding domain (DBD) within the hLigI catalytic fragment interacts with both PCNA and the heterotrimeric cell-cycle checkpoint clamp, hRad9-hRad1-hHus1 (9-1-1). The DBD preferentially binds to trimeric PCNA and the hRad1 subunit of 9-1-1. Unlike the majority of PCNA interacting proteins, the DBD does not interact with the interdomain connector loop region of PCNA but instead appears to interact with regions adjacent to the intersubunit interfaces within the PCNA trimer. Notably, the DBD not only binds specifically to DNA nicks but also mediates the formation of DNA protein complexes with PCNA. Based on these results, we suggest that the interface between the DBD and PCNA acts as a pivot facilitating the transition of the hLigI catalytic region fragment from an extended conformation to a ring structure when it engages a DNA nick. PMID:19523882

  12. Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro, is regulated by flap endonuclease 1 and DNA ligase 1.

    PubMed

    Crespan, Emmanuele; Hübscher, Ulrich; Maga, Giovanni

    2015-05-01

    Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.

  13. Transfection with replicating DNA from the temperate Bacillus bacteriophage phi 105 and with T4-ligase treated phi105 DNA: the importance in transfection of being longer than genome-length.

    PubMed

    Flock, J I

    1978-07-06

    Replicating phage DNA extracted from Bacillus subtilis infected with phage phi 105 has a higher activity in transfection than mature DNA. By heteroduplex analysis it was shown that this DNA contains concatemeric molecules. Concatemers, constructed in vitro by treatment of mature DNA with T4-ligase also have an increased activity in transfection. DNA showing an increased activity in transfection does not have a requirement for more than one molecule per transfection event as is typically found for transfection with mature phi 105 DNA. An explanation is given for this difference suggesting that the structure of the ends of the transfecting molecules play an important role intransfection.

  14. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

    PubMed Central

    Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

    2016-01-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  15. 4-coumarate:CoA ligase gene family in Rubus idaeus: cDNA structures, evolution, and expression.

    PubMed

    Kumar, Amrita; Ellis, Brian E

    2003-02-01

    The enzyme 4-coumarate:CoA ligase (4CL) activates cinnamic acid and its hydroxylated derivatives by forming the corresponding CoA thioesters. These serve as substrates for biosynthesis of phenylpropanoid-derived end-products that are important determinants of fruit quality in raspberry (Rubus idaeus L.). In higher plants, 4CL is typically encoded by a gene family. To investigate the participation of distinct 4CL genes in the process of fruit ripening, we have characterized this gene family in raspberry. By complementing a PCR-based homology search with low-stringency cDNA library screening, we have isolated three classes of raspberry 4CL cDNAs (Ri4CL1, Ri4CL2, and Ri4CL3). Phylogenetic analysis places the three raspberry 4CL gene family members into two distinct groups, a pattern consistent with an ancient divergence from an ancestral progenitor. Quantitative RT-PCR assay reveals a differential pattern of transcription of each of the three genes in various organs, as well as distinct temporal patterns of expression during flower and fruit development. The regulatory elements thus appear to have evolved independently of the genes themselves. Based on phylogenetic classification, expression patterns and recombinant protein activities the different Ri4CL genes are likely to participate in different biosynthetic pathways leading to the various phenylpropanoid-derived metabolites that help create flavor and color in raspberry fruit.

  16. Relationship between genetic polymorphisms of DNA ligase 1 and non-small cell lung cancer susceptibility and radiosensitivity.

    PubMed

    Tian, H; He, X; Yin, L; Guo, W J; Xia, Y Y; Jiang, Z X

    2015-06-26

    The aim of this study was to examine the relationship between genetic polymorphisms in DNA ligase 1 (LIG1) and non-small cell lung cancer (NSCLC) susceptibility and radiosensitivity in a Chinese population. This was a case-control study that included 352 NSCLC patients and 448 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was conducted to detect HaeIII polymorphisms in exon 6 of the LIG1 gene in this popula-tion. This information was used to observe the effects of radiation in pa-tients with different genotypes in order to determine the genotypes as-sociated with radiosensitivity. The CC genotype and C allele frequency were significantly higher in the NSCLC group than in the control group (P = 0.012 and P = 0.023, respectively). The relative risk of experienc-ing NSCLC was 2.55 [95% confidence interval (CI), 1.12-3.98] for CC homozygous patients and 0.87 (95%CI, 0.46-1.88) for AA homozygous patients. Analysis of LIG1 genetic polymorphisms and radiosensitiv-ity of NSCLC patients showed that AA homozygous patients were sig-nificantly more radiosensitive than the control group (AA vs AC, P = 0.014; AA vs CC, P < 0.001; AC vs CC, P = 0.023). Therefore, the LIG1 CC genotype was associated with susceptibility to NSCLC, and the AA genotype demonstrated increased radiosensitivity compared to the AC and CC genotypes.

  17. DNA Ligases I and III Cooperate in Alternative Non-Homologous End-Joining in Vertebrates

    PubMed Central

    Mladenov, Emil; Bencsik-Theilen, Alena; Bednar, Theresa; Wu, Wenqi; Arakawa, Hiroshi; Iliakis, George

    2013-01-01

    Biochemical and genetic studies suggest that vertebrates remove double-strand breaks (DSBs) from their genomes predominantly by two non-homologous end joining (NHEJ) pathways. While canonical NHEJ depends on the well characterized activities of DNA-dependent protein kinase (DNA-PK) and LIG4/XRCC4/XLF complexes, the activities and the mechanisms of the alternative, backup NHEJ are less well characterized. Notably, the contribution of LIG1 to alternative NHEJ remains conjectural and although biochemical, cytogenetic and genetic experiments implicate LIG3, this contribution has not been formally demonstrated. Here, we take advantage of the powerful genetics of the DT40 chicken B-cell system to delineate the roles of LIG1 and LIG3 in alternative NHEJ. Our results expand the functions of LIG1 to alternative NHEJ and demonstrate a remarkable ability for LIG3 to backup DSB repair by NHEJ in addition to its essential function in the mitochondria. Together with results on DNA replication, these observations uncover a remarkable and previously unappreciated functional flexibility and interchangeability between LIG1 and LIG3. PMID:23555685

  18. The tail that wags the dog: p12, the smallest subunit of DNA polymerase δ, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression.

    PubMed

    Lee, Marietta Y W T; Zhang, Sufang; Lin, Szu Hua Sharon; Wang, Xiaoxiao; Darzynkiewicz, Zbigniew; Zhang, Zhongtao; Lee, Ernest Y C

    2014-01-01

    DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4(Cdt2), participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4(Cdt2) partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4(Cdt2) now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.

  19. Dual-color detection of DNA sequence variants by ligase-mediated analysis

    SciTech Connect

    Samiotaki, M.; Kwiatkowski, M.; Parik, J.; Landegren, U. )

    1994-03-15

    Genetic screening for sequence variants associated with disease is assuming increasing importance in clinical medicine as well as in research. The authors describe an efficient method for such analyses, comprising a combination of practical features: (1) Amplified DNA samples are analyzed for their ability to serve as templates in standardized allele-specific ligation reactions between oligonucleotide probes; (2) Two allele-specific probes, differentially labeled with either of two lanthanide labels, compete for ligation to a third oligonucleotide (the signal from the two labeled probes can thus be directly compared in a sensitive time-resolved fluorescence detection reaction); and (3) Large sets of analyses are processed in parallel using a 96-pin capture manifold, serving to reduce pipetting steps and the risk of contamination. The authors present here the basis of the technique and its application to the screening for two common mutations causing cystic fibrosis and [alpha][sub 1]-antiytrypsin deficiency. 19 refs., 4 figs.

  20. Homology Modeling of NAD+-Dependent DNA Ligase of the Wolbachia Endosymbiont of Brugia malayi and Its Drug Target Potential Using Dispiro-Cycloalkanones

    PubMed Central

    Shrivastava, Nidhi; Nag, Jeetendra K.; Pandey, Jyoti; Tripathi, Rama Pati; Shah, Priyanka; Siddiqi, Mohammad Imran

    2015-01-01

    Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD+-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD+-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD+ cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 μM), followed by compound 5 (IC50, 2.3 μM) and compound 1 (IC50, 2.9 μM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates. PMID:25845868

  1. Homology modeling of NAD+-dependent DNA ligase of the Wolbachia endosymbiont of Brugia malayi and its drug target potential using dispiro-cycloalkanones.

    PubMed

    Shrivastava, Nidhi; Nag, Jeetendra K; Pandey, Jyoti; Tripathi, Rama Pati; Shah, Priyanka; Siddiqi, Mohammad Imran; Misra-Bhattacharya, Shailja

    2015-07-01

    Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD(+)-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD(+)-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD(+) cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 μM), followed by compound 5 (IC50, 2.3 μM) and compound 1 (IC50, 2.9 μM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

  2. Targeting Breast Cancer with 4 Steroid Adapter to Inhibit DNA Repair

    DTIC Science & Technology

    2012-01-01

    efficient assay capable of detecting assembly of the core ligase complex involved in nonhomologous end joining ( NHEJ ) of DNA double-strand breaks. The...synthesis of the adapter. 15. SUBJECT TERMS DNA repair, molecular adapter, nonhomologous end joining 16. SECURITY CLASSIFICATION OF 17...S.P. (2006). XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end - joining . Cell 124, 301-313. DeFazio, L., Stansel, R

  3. Inhibiting NAD+-dependent DNA ligase activity with 2-(cyclopentyloxy)-5'-deoxyadenosine (CPOdA) offers a new tool for DNA replication and repair studies in the model archaeon Haloferax volcanii.

    PubMed

    Giroux, Xavier; MacNeill, Stuart A

    2015-11-01

    DNA ligases play an essential role in many aspects of DNA metabolism in all three domains of life. The haloarchaeal organism Haloferax volcanii encodes both ATP- and NAD(+)-dependent DNA ligase enzymes designated LigA and LigN, respectively. Neither LigA nor LigN alone is required for cell viability but they share an essential function, most likely the ligation of Okazaki fragments during chromosome replication. Here we show that 2-(cyclopentyloxy)-5'-deoxyadenosine (referred to as CPOdA), originally developed as a inhibitor of bacterial NAD(+)-dependent DNA ligases, is a potent inhibitor of the growth of Hfx. volcanii cells expressing LigN alone, causing chromosome fragmentation and cell death, while cells expressing LigA are unaffected. Growth inhibition occurs at significantly lower CPOdA concentrations (MIC ≤ 50 ng ml(-1)) than those required for inhibition of bacterial growth (≥2 μg ml(-1)). CPOdA has the potential to become a vital tool in DNA replication and repair studies in this important model organism.

  4. The RuvAB branch migration complex can displace topoisomerase IV.quinolone.DNA ternary complexes.

    PubMed

    Shea, Molly E; Hiasa, Hiroshi

    2003-11-28

    Quinolone antimicrobial drugs target both DNA gyrase and topoisomerase IV (Topo IV) and convert these essential enzymes into cellular poisons. Topoisomerase poisoning results in the inhibition of DNA replication and the generation of double-strand breaks. Double-strand breaks are repaired by homologous recombination. Here, we have investigated the interaction between the RuvAB branch migration complex and the Topo IV.quinolone.DNA ternary complex. A strand-displacement assay is employed to assess the helicase activity of the RuvAB complex in vitro. RuvAB-catalyzed strand displacement requires both RuvA and RuvB proteins, and it is stimulated by a 3'-non-hybridized tail. Interestingly, Topo IV.quinolone.DNA ternary complexes do not inhibit the translocation of the RuvAB complex. In fact, Topo IV.quinolone.DNA ternary complexes are reversed and displaced from the DNA upon their collisions with the RuvAB complex. These results suggest that the RuvAB branch migration complex can actively remove quinolone-induced covalent topoisomerase.DNA complexes from DNA and complete the homologous recombination process in vivo.

  5. Synthesis, Acetylation, and Phosphorylation of Histone IV and Its Binding to DNA During Spermatogenesis in Trout*

    PubMed Central

    Louie, Andrew J.; Dixon, Gordon H.

    1972-01-01

    During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [3H]aminoacids and inorganic [32P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [3H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [3H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total 3H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an “obligatory” acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that ε-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH2-terminal region of histone IV to assume the correct conformation (in this case, an α-helix), and fit into the major groove of DNA. Deacetylation then “locks” histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known. Images PMID:4505675

  6. A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the "Coffee Break Ligation" technique.

    PubMed

    Yoshino, Yuki; Ishida, Masaharu; Horii, Akira

    2007-10-01

    The ligation reaction is widely used in molecular biology. There are several kits available that complete the ligation reaction very rapidly but they are rather expensive. In this study, we successfully modified the ligation buffer with much lower cost than existing kits. The ligation reaction can be completed in 10 min using very low activities such as 0.01 U T4 DNA ligase, and costs only $1 for 100 reactions of 20 microl scale. We name this ligation system the "Coffee Break Ligation" system; one can complete ligation reaction while drinking a cup of coffee, and perform 100 reactions by spending money equivalent to a cup of coffee.

  7. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

    PubMed Central

    Mohiuddin; Kobayashi, Shunsuke; Keka, Islam Shamima; Guilbaud, Guillaume; Sale, Julian; Narita, Takeo; Abdel-Aziz, H. Ismail; Wang, Xin; Ogawa, Saki; Sasanuma, Hiroyuki; Chiu, Roland; Oestergaard, Vibe H.; Lisby, Michael; Takeda, Shunichi

    2017-01-01

    The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2-/- and RNF8-/- cells and HERC2-/-/RNF8-/- double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2-/- and RNF8-/- mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks. PMID:26994443

  8. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line.

    PubMed

    Mohiuddin; Kobayashi, Shunsuke; Keka, Islam Shamima; Guilbaud, Guillaume; Sale, Julian; Narita, Takeo; Abdel-Aziz, H Ismail; Wang, Xin; Ogawa, Saki; Sasanuma, Hiroyuki; Chiu, Roland; Oestergaard, Vibe H; Lisby, Michael; Takeda, Shunichi

    2016-04-01

    The replicative DNA polymerases are generally blocked by template DNA damage. The resulting replication arrest can be released by one of two post-replication repair (PRR) pathways, translesion DNA synthesis (TLS) and template switching by homologous recombination (HR). The HERC2 ubiquitin ligase plays a role in homologous recombination by facilitating the assembly of the Ubc13 ubiquitin-conjugating enzyme with the RNF8 ubiquitin ligase. To explore the role of HERC2 and RNF8 in PRR, we examined immunoglobulin diversification in chicken DT40 cells deficient in HERC2 and RNF8. Unexpectedly, the HERC2(-/-) and RNF8(-/-) cells and HERC2(-/-)/RNF8(-/-) double mutant cells exhibit a significant reduction in the rate of immunoglobulin (Ig) hypermutation, compared to wild-type cells. Further, the HERC2(-/-) and RNF8(-/-) mutants exhibit defective maintenance of replication fork progression immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks.

  9. DNA Cleavage Activities of Staphylococcus aureus Gyrase and Topoisomerase IV Stimulated by Quinolones and 2-Pyridones

    PubMed Central

    Saiki, Anne Y. C.; Shen, Linus L.; Chen, Chih-Ming; Baranowski, John; Lerner, Claude G.

    1999-01-01

    We have cloned Staphylococcus aureus DNA gyrase and topoisomerase IV and expressed them in Escherichia coli as polyhistidine-tagged proteins to facilitate purification and eliminate contamination by host enzymes. The enzyme preparations had specific activities similar to previously reported values. Potassium glutamate (K-Glu) stimulated the drug-induced DNA cleavage activity and was optimal between 100 and 200 mM for gyrase and peaked at 100 mM for topoisomerase IV. Higher concentrations of K-Glu inhibited the cleavage activities of both enzymes. Using a common buffer system containing 100 mM K-Glu, we tested the enzyme-mediated DNA cleavage activities of both gyrase and topoisomerase IV with oxolinic acid, norfloxacin, ciprofloxacin, trovafloxacin, clinafloxacin, and the 2-pyridone ABT-719. As expected, all drugs tested demonstrated greater potency against topoisomerase IV than against gyrase. In addition, cleavage activity was found to correlate well with antibacterial activity. PMID:10390205

  10. The diagonal hopping dependent I-V characteristics of periodic DNA molecule

    NASA Astrophysics Data System (ADS)

    Yudiarsah, E.

    2017-07-01

    The diagonal hopping dependent I-V characteristic of a periodic DNA molecule has been studied. Exponentially twisting angle dependent hopping constant is used in the calculation. The diagonal hopping constant is modeled using semi-empirical Slater-Koster theory. A 32 base pairs doubled-stranded Poly(dG)-poly(dC) DNA molecule model sandwiched in between two metallic electrodes is used in the calculation of I-V characteristic using Landauer-Buttiker formalism by assuming symmetric voltage drop at the contacts. The I-V characteristics is calculated from the transmission probability of charge on the molecule calculated using transfer and scattering matrix methods, simultaneously. The results show that generally, as the vibration and diagonal hopping coupling constant increases, the current magnitude increases. However, at low voltage the increment of current is minute. These trends are observed in the I-V characteristic of DNA molecule for all frequencies and temperatures used.

  11. 3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

    PubMed

    Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

    2017-09-22

    5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Vanadium(IV) and copper(II) complexes of salicylaldimines and aromatic heterocycles: Cytotoxicity, DNA binding and DNA cleavage properties.

    PubMed

    Correia, Isabel; Roy, Somnath; Matos, Cristina P; Borovic, Sladjana; Butenko, Nataliya; Cavaco, Isabel; Marques, Fernanda; Lorenzo, Julia; Rodríguez, Alejandra; Moreno, Virtudes; Pessoa, João Costa

    2015-06-01

    Five copper(II) complexes, [Cu(sal-Gly)(bipy)](1), [Cu(sal-Gly)(phen)] (2), [Cu(sal-l-Ala)(phen)] (3), [Cu(sal-D-Ala)(phen)] (4), [Cu(sal-l-Phe)(phen)] (5) and five oxidovanadium(IV) complexes, [V(IV)O(sal-Gly)(bipy)] (6), [V(IV)O(sal-Gly)(phen)] (7), [V(IV)O(sal-l-Phe)(H2O)] (8), [V(IV)O(sal-l-Phe)(bipy)] (9), [V(IV)O(sal-l-Phe)(phen)] (10) (sal=salicylaldehyde, bipy=2,2'-bipyridine, phen=1,10-phenanthroline) were synthesized and characterized, and their interaction with DNA was evaluated by different techniques: gel electrophoresis, fluorescence, UV-visible and circular dichroism spectroscopy. The complexes interact with calf-thymus DNA and efficiently cleave plasmid DNA in the absence (only 2 and 5) and/or presence of additives. The cleavage ability is concentration-dependent as well as metal and ligand-dependent. Moreover, DNA binding experiments show that the phen-containing Cu(II) and V(IV)O compounds display stronger DNA interaction ability than the corresponding bipy analogues. The complexes present cytotoxic activity against human ovarian (A2780) and breast (MCF7) carcinoma cells. Cell-growth inhibition (IC50) of compounds 1, 2 and 5 in human promyelocytic leukemia (HL60) and human cervical cancer (HeLa) cells were also determined. The copper complexes show much higher cytotoxic activity than the corresponding vanadium complexes and the reference drug cisplatin (except for the sal-Gly complexes); namely, the phenanthroline copper complexes 2-5 are ca. 10-fold more cytotoxic than cisplatin and more cytotoxic than their bipyridine analogues.

  13. Screen for abnormal mitochondrial phenotypes in mouse embryonic stem cells identifies a model for succinyl-CoA ligase deficiency and mtDNA depletion

    PubMed Central

    Donti, Taraka R.; Stromberger, Carmen; Ge, Ming; Eldin, Karen W.; Craigen, William J.; Graham, Brett H.

    2014-01-01

    ABSTRACT Mutations in subunits of succinyl-CoA synthetase/ligase (SCS), a component of the citric acid cycle, are associated with mitochondrial encephalomyopathy, elevation of methylmalonic acid (MMA), and mitochondrial DNA (mtDNA) depletion. A FACS-based retroviral-mediated gene trap mutagenesis screen in mouse embryonic stem (ES) cells for abnormal mitochondrial phenotypes identified a gene trap allele of Sucla2 (Sucla2SAβgeo), which was used to generate transgenic mice. Sucla2 encodes the ADP-specific β-subunit isoform of SCS. Sucla2SAβgeo homozygotes exhibited recessive lethality, with most mutants dying late in gestation (e18.5). Mutant placenta and embryonic (e17.5) brain, heart and muscle showed varying degrees of mtDNA depletion (20–60%). However, there was no mtDNA depletion in mutant liver, where the gene is not normally expressed. Elevated levels of MMA were observed in embryonic brain. SCS-deficient mouse embryonic fibroblasts (MEFs) demonstrated a 50% reduction in mtDNA content compared with wild-type MEFs. The mtDNA depletion resulted in reduced steady state levels of mtDNA encoded proteins and multiple respiratory chain deficiencies. mtDNA content could be restored by reintroduction of Sucla2. This mouse model of SCS deficiency and mtDNA depletion promises to provide insights into the pathogenesis of mitochondrial diseases with mtDNA depletion and into the biology of mtDNA maintenance. In addition, this report demonstrates the power of a genetic screen that combines gene trap mutagenesis and FACS analysis in mouse ES cells to identify mitochondrial phenotypes and to develop animal models of mitochondrial dysfunction. PMID:24271779

  14. Construction of a cDNA clone corresponding to mouse alpha 1(IV) procollagen.

    PubMed Central

    dos Santos, C L; Villa, L L; Sonohara, S; Brentani, R R

    1984-01-01

    A new procedure for the synthesis of double stranded cDNA, based upon release of mRNA by "in vitro" translation, was used to clone type IV collagen. Collagen synthesizing polysomes selectively isolated from a mouse parietal yolk sac carcinoma (PYS-2) were used for translation in an heterologous cell-free system. Translation products were collagenase-sensitive and displayed an electrophoretic mobility correspondent to type IV collagen. Translation released mRNA was employed to construct a 100 base pairs long cDNA clone which hybridized to a 7,800 nucleotides long mRNA. Peptides synthesized by "in vitro" translation of hybrid selected mRNA displayed an electrophoretic mobility compatible with that of alpha 1 (IV) collagen, were sensitive to collagenase and were immunoprecipitated by anti-type IV collagen antibody. Images PMID:6546618

  15. Contribution of Topoisomerase IV and DNA Gyrase Mutations in Streptococcus pneumoniae to Resistance to Novel Fluoroquinolones

    PubMed Central

    Pestova, Ekaterina; Beyer, Rebecca; Cianciotto, Nicholas P.; Noskin, Gary A.; Peterson, Lance R.

    1999-01-01

    In this study, we assessed the activity of ciprofloxacin, levofloxacin, sparfloxacin, and trovafloxacin against clinical isolates of Streptococcus pneumoniae that were resistant to the less-recently developed fluoroquinolones by using defined amino acid substitutions in DNA gyrase and topoisomerase IV. The molecular basis for resistance was assessed by using mutants selected with trovafloxacin, ciprofloxacin, and levofloxacin in vitro. This demonstrated that the primary target of trovafloxacin in S. pneumoniae is the ParC subunit of DNA topoisomerase IV, similar to most other fluoroquinolones. However, first-step mutants bearing the Ser79→Phe/Tyr substitution in topoisomerase IV subunit ParC were susceptible to trovafloxacin with a minimum inhibitory concentration of 0.25 μg/ml, and mutations in the structural genes for both topoisomerase IV subunit ParC (parC) and the DNA gyrase subunit (gyrA) were required to achieve levels of resistance above the breakpoint. The data also suggest that enhanced activity of trovafloxacin against pneumococci is due to a combination of factors that may include reduced efflux of this agent and an enhanced activity against both DNA gyrase and topoisomerase IV. PMID:10428926

  16. Partial suppression of bacteriophage T4 ligase mutations by T4 endonuclease II deficiency: role of host ligase.

    PubMed

    Warner, H R

    1971-04-01

    Endonuclease II-deficient, ligase-deficient double mutants of phage T4 induce considerably more deoxyribonucleic acid (DNA) synthesis after infection of Escherichia coli B than does the ligase-deficient single mutant. Furthermore, the double mutant can replicate 10 to 15% as well as wild-type T4, whereas the single mutant fails to replicate. When the E. coli host is also deficient in ligase, the double mutant resembles the single mutant. The results indicate that host ligase can substitute for phage ligase when the host DNA is not attacked by the phage-induced endonuclease II.

  17. DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity.

    PubMed

    East, Stephen P; White, Clara Bantry; Barker, Oliver; Barker, Stephanie; Bennett, James; Brown, David; Boyd, E Andrew; Brennan, Christopher; Chowdhury, Chandana; Collins, Ian; Convers-Reignier, Emmanuelle; Dymock, Brian W; Fletcher, Rowena; Haydon, David J; Gardiner, Mihaly; Hatcher, Stuart; Ingram, Peter; Lancett, Paul; Mortenson, Paul; Papadopoulos, Konstantinos; Smee, Carol; Thomaides-Brears, Helena B; Tye, Heather; Workman, James; Czaplewski, Lloyd G

    2009-02-01

    The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.

  18. Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

    PubMed Central

    Pitts, Steven L.; Liou, Grace F.; Mitchenall, Lesley A.; Burgin, Alex B.; Maxwell, Anthony; Neuman, Keir C.; Osheroff, Neil

    2011-01-01

    It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme. PMID:21300644

  19. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD(+)-dependent DNA ligase inhibitor.

    PubMed

    Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

    2016-09-01

    Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria.

  20. The carboxyl terminus of FANCE recruits FANCD2 to the Fanconi Anemia (FA) E3 ligase complex to promote the FA DNA repair pathway.

    PubMed

    Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D; Kee, Younghoon

    2014-03-07

    Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair.

  1. The Carboxyl Terminus of FANCE Recruits FANCD2 to the Fanconi Anemia (FA) E3 Ligase Complex to Promote the FA DNA Repair Pathway*

    PubMed Central

    Polito, David; Cukras, Scott; Wang, Xiaozhe; Spence, Paige; Moreau, Lisa; D'Andrea, Alan D.; Kee, Younghoon

    2014-01-01

    Fanconi anemia (FA) is a genome instability syndrome characterized by bone marrow failure and cellular hypersensitivity to DNA cross-linking agents. In response to DNA damage, the FA pathway is activated through the cooperation of 16 FA proteins. A central player in the pathway is a multisubunit E3 ubiquitin ligase complex or the FA core complex, which monoubiquitinates its substrates FANCD2 and FANCI. FANCE, a subunit of the FA core complex, plays an essential role by promoting the integrity of the complex and by directly recognizing FANCD2. To delineate its role in substrate ubiquitination from the core complex assembly, we analyzed a series of mutations within FANCE. We report that a phenylalanine located at the highly conserved extreme C terminus, referred to as Phe-522, is a critical residue for mediating the monoubiquitination of the FANCD2-FANCI complex. Using the FANCE mutant that specifically disrupts the FANCE-FANCD2 interaction as a tool, we found that the interaction-deficient mutant conferred cellular sensitivity in reconstituted FANCE-deficient cells to a similar degree as FANCE null cells, suggesting the significance of the FANCE-FANCD2 interaction in promoting cisplatin resistance. Intriguingly, ectopic expression of the FANCE C terminus fragment alone in FA normal cells disrupts DNA repair, consolidating the importance of the FANCE-FANCD2 interaction in the DNA cross-link repair. PMID:24451376

  2. Differential response between the p53 ubiquitin-protein ligases Pirh2 and MdM2 following DNA damage in human cancer cells

    SciTech Connect

    Duan Wenrui; Gao, Li; Wu Xin; Zhang Yang; Otterson, Gregory A.; Villalona-Calero, Miguel A. . E-mail: Miguel.villalona@osumc.edu

    2006-10-15

    Pirh2, a recently identified ubiquitin-protein ligase, has been reported to promote p53 degradation. Pirh2 physically interacts with p53 and promotes ubiquitination of p53 independently of MDM2. Like MDM2, Pirh2 is thought to participate in an autoregulatory feedback loop that controls p53 function. We have previously reported that Pirh2 was overexpressed in human and murine lung cancers as compared to uninvolved lung tissue. Pirh2 increase could potentially cause degradation of wildtype p53 and reduce its tumor suppression function in the lung tumor cells. Since Pirh2 has been reported to be transactivated by p53, however, the mechanisms by which a high level of Pirh2 expression is maintained in tumor cells despite low level of wildtype p53 protein are unclear. In order to evaluate p53 involvement in the transactivation of Pirh2, we evaluated Pirh2, MDM2, p53 and p21 expression with Western blot analysis and real time PCR after {gamma} irradiation or cisplatin DNA damage treatment using human cancer cell lines containing wildtype (A549, MCF-7), mutant (H719) and null (H1299) p53. Surprisingly, Pirh2 expression was not affected by the presence of wildtype p53 in the cancer cells. In contrast, MDM2 was upregulated by wildtype p53 in A549 and MCF-7 cells and was absent from the H1299 and the H719 cells. We conclude that Pirh2 operates in a distinct manner from MDM2 in response to DNA damage in cancer cells. Pirh2 elevation in p53 null cells indicates the existence of additional molecular mechanisms for Pirh2 upregulation and suggests that p53 is not the sole target of Pirh2 ubiquitin ligase activity.

  3. The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms

    PubMed Central

    White, Travis B.; Lambowitz, Alan M.

    2012-01-01

    Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential

  4. Inhibition of DNA gyrase and DNA topoisomerase IV of Staphylococcus aureus and Escherichia coli by aminocoumarin antibiotics.

    PubMed

    Alt, Silke; Mitchenall, Lesley A; Maxwell, Anthony; Heide, Lutz

    2011-09-01

    Aminocoumarin antibiotics are potent inhibitors of bacterial DNA gyrase. We investigated the inhibitory and antibacterial activity of naturally occurring aminocoumarin antibiotics and six structural analogues (novclobiocins) against DNA gyrase and DNA topoisomerase IV from Escherichia coli and Staphylococcus aureus as well as the effect of potassium and sodium glutamate on the activity of these enzymes. The inhibitory concentrations of the aminocoumarins were determined in gyrase supercoiling assays and topoisomerase IV decatenation assays. Both subunits of S. aureus topoisomerase IV were purified as His-Tag proteins in E. coli. The MIC was tested in vivo for the control organisms E. coli ATCC 25922 and S. aureus ATCC 29213. DNA gyrase is the primary target in vitro of all investigated aminocoumarins. With the exception of simocyclinone D8, all other aminocoumarins inhibited S. aureus gyrase on average 6-fold more effectively than E. coli gyrase. Potassium glutamate is essential for the activity of S. aureus gyrase and increases the sensitivity of E. coli gyrase to aminocoumarins ≥ 10-fold. The antibacterial activity of the tested compounds mirrored their relative activities against topoisomerases. The study provides insights about the substituents that are important for the inhibitory activity of aminocoumarins against the target enzymes, which will facilitate the rational design of improved antibiotics.

  5. The H2B ubiquitin ligase RNF40 cooperates with SUPT16H to induce dynamic changes in chromatin structure during DNA double-strand break repair.

    PubMed

    Kari, Vijayalakshmi; Shchebet, Andrei; Neumann, Heinz; Johnsen, Steven A

    2011-10-15

    Many anticancer therapies function largely by inducing DNA double-strand breaks (DSBs) or altering the ability of cancer cells to repair them. Proper and timely DNA repair requires dynamic changes in chromatin assembly and disassembly characterized by histone H3 lysine 56 acetylation (H3K56ac) and phosphorylation of the variant histone H2AX (γH2AX). Similarly, histone H2B monoubiquitination (H2Bub1) functions in DNA repair, but its role in controlling dynamic changes in chromatin structure following DSBs and the histone chaperone complexes involved remain unknown. Therefore, we investigated the role of the H2B ubiquitin ligase RNF40 in the DSB response. We show that RNF40 depletion results in sustained H2AX phosphorylation and a decrease in rapid cell cycle checkpoint activation. Furthermore, RNF40 knockdown resulted in decreased H3K56ac and decreased recruitment of the facilitates chromatin transcription (FACT) complex to chromatin following DSB. Knockdown of the FACT component suppressor of Ty homolog-16 (SUPT16H) phenocopied the effects of RNF40 knockdown on both γH2AX and H3K56ac following DSB induction. Consistently, both RNF40 and SUPT16H were required for proper DNA end resection and timely DNA repair, suggesting that H2Bub1 and FACT cooperate to increase chromatin dynamics, which facilitates proper checkpoint activation and timely DNA repair. These results provide important mechanistic insights into the tumor suppressor function of H2Bub1 and provide a rational basis for pursuing H2Bub1-based therapies in conjunction with traditional chemo- and radiotherapy.

  6. DNA cleavage activity of V IV O(acac)2 and derivatives.

    PubMed

    Butenko, Nataliya; Tomaz, Ana Isabel; Nouri, Ofelia; Escribano, Esther; Moreno, Virtudes; Gama, Sofia; Ribeiro, Vera; Telo, João Paulo; Pesssoa, João Costa; Cavaco, Isabel

    2009-04-01

    The DNA cleavage activity of several beta-diketonate vanadyl complexes is examined. Vanadyl acetylacetonate, V(IV)O(acac)(2), 1, shows a remarkable activity in degrading plasmid DNA in the absence of any activating agents, air and photoirradiation. The cleaving activity of several related complexes V(IV)O(hd)(2) (2, Hhd=3,5-heptanedione), V(IV)O(acac-NH(2))(2) (3, Hacac-NH(2)=acetoacetamide) and V(IV)O(acac-NMe(2))(2) (4, Hacac-NMe(2)=N,N-dimethylacetoacetamide) is also evaluated. It is shown that 2 exhibits an activity similar to 1, while 3 and 4 are much less efficient cleaving agents. The different activity of the complexes is related to their stability towards hydrolysis in aqueous solution, which follows the order 1 approximately 2>3 approximately 4. The nature of the pH buffer was also found to be determinant in the nuclease activity of 1 and 2. In a phosphate buffered medium DNA cleavage by these agents is much more efficient than in tris, hepes, mes or mops buffers. The reaction seems to take place through a mixed mechanism, involving the formation of reactive oxygen species (ROS), namely OH radicals, and possibly also direct cleavage at phosphodiester linkages induced by the vanadium complexes.

  7. Septic patients with mitochondrial DNA haplogroup JT have higher respiratory complex IV activity and survival rate.

    PubMed

    Lorente, Leonardo; Martín, María M; López-Gallardo, Ester; Ferreres, José; Solé-Violán, Jordi; Labarta, Lorenzo; Díaz, César; Jiménez, Alejandro; Montoya, Julio; Ruiz-Pesini, Eduardo

    2016-06-01

    The influence of mitochondrial deoxyribonucleic acid (mtDNA) haplogroup or oxidative phosphorylation system (OXPHOS) function on survival of septic patients has been scarcely studied. However, the association between mtDNA haplogroup, OXPHOS capacity at diagnosis of severe sepsis, and survival has been not previously reported, and that was the objective of the present study. This was a prospective, multicenter, observational study. Blood samples from 198 patients at diagnosis of severe sepsis were analyzed to determine mtDNA haplogroup and platelet respiratory complex IV (CIV) specific activity. The end point of the study was 30-day survival. Septic patients with mtDNA haplogroup JT showed higher 30-day survival than those with mtDNA haplogroup non-JT (31/38 [81.6%] vs 99/160 [61.9%]; P= .02). Septic patients with mtDNA haplogroup JT showed higher platelet CIV specific activity than those with mtDNA haplogroup non-JT (P= .002). The main novel finding of our study, including the largest series providing data on platelet CIV specific activity according to mtDNA haplogroup in severe septic patients, was that those with mtDNA haplogroup JT showed higher survival and higher platelet CIV specific activity at diagnosis of severe sepsis than patients with mtDNA haplogroup non-JT. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

    PubMed Central

    Pan, Xiao-Su; Fisher, L. Mark

    1999-01-01

    Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic

  9. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation.

    PubMed

    Rawdon, Eric J; Dorier, Julien; Racko, Dusan; Millett, Kenneth C; Stasiak, Andrzej

    2016-06-02

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.

  10. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

    PubMed Central

    Rawdon, Eric J.; Dorier, Julien; Racko, Dusan; Millett, Kenneth C.; Stasiak, Andrzej

    2016-01-01

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation. PMID:27106058

  11. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2002-07-01

    DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as other DNA repair components including NBS 1 and MRE11. In contrast to the original report, 90% of the epithelial cells in normal

  12. Differential Expression of DNA Double-Strand Break Repair Proteins in Breast Cells

    DTIC Science & Technology

    2003-07-01

    DNA-PK in human breast tissues by immuno-histochemistry and extended these studies to two other components of the NHEJ repair pathway, XRCC4 and DNA ... ligase IV, as well as other DNA repair components including NBSl and MRE11. In contrast to the original report, 90% of the epithelial cells in normal

  13. SOS mutator DNA polymerase IV functions in adaptive mutation and not adaptive amplification.

    PubMed

    McKenzie, G J; Lee, P L; Lombardo, M J; Hastings, P J; Rosenberg, S M

    2001-03-01

    Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

  14. Clerocidin interacts with the cleavage complex of Streptococcus pneumoniae topoisomerase IV to induce selective irreversible DNA damage.

    PubMed

    Richter, Sara N; Leo, Elisabetta; Giaretta, Giulia; Gatto, Barbara; Fisher, L Mark; Palumbo, Manlio

    2006-01-01

    Clerocidin (CL), a diterpenoid natural product, alkylates DNA through its epoxide moiety and exhibits both anticancer and antibacterial activities. We have examined CL action in the presence of topoisomerase IV from Streptococcus pneumoniae. CL promoted irreversible enzyme-mediated DNA cleavage leading to single- and double-stranded DNA breaks at specific sites. Reaction required the diterpenoid function: no cleavage was seen using a naphthalene-substituted analogue. Moreover, drug-induced DNA breakage was not observed using a mutant topoisomerase IV (ParC Y118F) unable to form a cleavage complex with DNA. Sequence analysis of 102 single-stranded DNA breaks and 79 double-stranded breaks revealed an overwhelming preference for G at the -1 position, i.e. immediately 5' of the enzyme DNA scission site. This specificity contrasts with that of topoisomerase IV cleavage with antibacterial quinolones. Indeed, CL stimulated DNA breakage by a quinolone-resistant topoisomerase IV (ParC S79F). Overall, the results indicate that topoisomerase IV facilitates selective irreversible CL attack at guanine and that its cleavage complex differs markedly from that of mammalian topoisomerase II which promotes both irreversible and reversible CL attack at guanine and cytosine, respectively. The unique ability to form exclusively irreversible DNA breaks suggests topoisomerase IV may be a key intracellular target of CL in bacteria.

  15. Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.

    PubMed

    Muth, J; Williams, P M; Williams, S J; Brown, M D; Wallace, D C; Karger, B L

    1996-12-01

    High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.

  16. Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining.

    PubMed

    Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E; Iliakis, George

    2014-06-01

    In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Requirement for Parp-1 and DNA ligases 1 or 3 but not of Xrcc1 in chromosomal translocation formation by backup end joining

    PubMed Central

    Soni, Aashish; Siemann, Maria; Grabos, Martha; Murmann, Tamara; Pantelias, Gabriel E.; Iliakis, George

    2014-01-01

    In mammalian cells, ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) are repaired in all phases of the cell cycle predominantly by classical, DNA-PK-dependent nonhomologous end joining (D-NHEJ). Homologous recombination repair (HRR) is functional during the S- and G2-phases, when a sister chromatid becomes available. An error-prone, alternative form of end joining, operating as backup (B-NHEJ) functions robustly throughout the cell cycle and particularly in the G2-phase and is thought to backup predominantly D-NHEJ. Parp-1, DNA-ligases 1 (Lig1) and 3 (Lig3), and Xrcc1 are implicated in B-NHEJ. Chromosome and chromatid translocations are manifestations of erroneous DSB repair and are crucial culprits in malignant transformation and IR-induced cell lethality. We analyzed shifts in translocation formation deriving from defects in D-NHEJ or HRR in cells irradiated in the G2-phase and identify B-NHEJ as the main DSB repair pathway backing up both of these defects at the cost of a large increase in translocation formation. Our results identify Parp-1 and Lig1 and 3 as factors involved in translocation formation and show that Xrcc1 reinforces the function of Lig3 in the process without being required for it. Finally, we demonstrate intriguing connections between B-NHEJ and DNA end resection in translocation formation and show that, as for D-NHEJ and HRR, the function of B-NHEJ facilitates the recovery from the G2-checkpoint. These observations advance our understanding of chromosome aberration formation and have implications for the mechanism of action of Parp inhibitors. PMID:24748665

  18. Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase*S⃞

    PubMed Central

    Wang, Li Kai; Nair, Pravin A.; Shuman, Stewart

    2008-01-01

    NAD+-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg333); penetrate the minor grove and distort the nick (Val383 and Ile384); or stabilize the OB fold (Arg379). The essential constituents of the HhH domain include: four glycines (Gly455, Gly489, Gly521, Gly553), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg487, which penetrates the minor groove at the outer margin on the 3 ®-OH side of the nick; and Arg446, which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation. PMID:18515356

  19. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology

    PubMed Central

    Chambers, Cecilia R.; Patrick, Wayne M.

    2015-01-01

    With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area. PMID:26494982

  20. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology.

    PubMed

    Chambers, Cecilia R; Patrick, Wayne M

    2015-01-01

    With their ability to catalyse the formation of phosphodiester linkages, DNA ligases and RNA ligases are essential tools for many protocols in molecular biology and biotechnology. Currently, the nucleic acid ligases from bacteriophage T4 are used extensively in these protocols. In this review, we argue that the nucleic acid ligases from Archaea represent a largely untapped pool of enzymes with diverse and potentially favourable properties for new and emerging biotechnological applications. We summarise the current state of knowledge on archaeal DNA and RNA ligases, which makes apparent the relative scarcity of information on in vitro activities that are of most relevance to biotechnologists (such as the ability to join blunt- or cohesive-ended, double-stranded DNA fragments). We highlight the existing biotechnological applications of archaeal DNA ligases and RNA ligases. Finally, we draw attention to recent experiments in which protein engineering was used to modify the activities of the DNA ligase from Pyrococcus furiosus and the RNA ligase from Methanothermobacter thermautotrophicus, thus demonstrating the potential for further work in this area.

  1. Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli.

    PubMed

    Silverman, Adam P; Jiang, Qingfei; Goodman, Myron F; Kool, Eric T

    2007-12-04

    The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.

  2. Anticancer and DNA binding activities of platinum (IV) complexes; importance of leaving group departure rate.

    PubMed

    Pouryasin, Zahra; Yousefi, Reza; Nabavizadeh, S Masoud; Rashidi, Mehdi; Hamidizadeh, Peyman; Alavianmehr, Mohammad-Mehdi; Moosavi-Movahedi, Ali Akbar

    2014-03-01

    The two six-coordinate Pt(IV) complexes, containing bidentate nitrogen donor/methyl ligands with general formula [Pt(X)2Me2((t)bu2bpy)], where (t)bu2bpy = 4,4'-ditert-butyl-2,2'-bipyridine and X = Cl (C1) or Br (C2), serving as the leaving groups were synthesized for evaluation of their anticancer activities and DNA binding properties. To examine anticancer activities of the synthetic complexes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ethidium bromide/acridine orange (EB/AO) staining method were performed. The binding properties of these complexes to DNA and purine nucleotides were examined, using different spectroscopic techniques. These complexes demonstrated significant anticancer activities against three cancer cell lines Jurkat, K562, and MCF-7. On the basis of the results of EB/AO staining, C1 and C2 were also capable to induce apoptosis in cancer cells. These complexes comprise halide leaving groups, displaying different departure rates; accordingly, they demonstrated slightly dissimilar anticancer activity and significantly different DNA/purine nucleotide binding properties. The results of DNA interaction studies of these complexes suggest a mixed-binding mode, comprising partial intercalation and groove binding. Overall, the results presented herein indicate that the newly synthesized Pt(IV) complexes are promising class of the potential anticancer agents which can be considered as molecular templates in designing novel platinum anticancer drugs. This study also highlights the importance of leaving group in anticancer activity and DNA binding properties of Pt(IV) complexes.

  3. Poly (ADP-Ribose) Polymerase (PARP) is Essential for Sulfur Mustard-Induced DNA Damage Repair, But Has No Role in DNA Ligase Activation

    DTIC Science & Technology

    2006-01-01

    ligase activation could be due to its modification by PARP. Using HEK, intracellular "H-labeled NAD÷ (H-adenine) was metabolically generated and then... acetic acid methyl ester) (Bhat et al., 1998). These observations indicate a Stock HEK from adult skin of a single donor at passage need for a better...0.76 HEK were used in which NAD’ was metabolically 3H- Z-VAD-FMK (4 pm) 0.55CDO5 antibodly (2 pag ml )/(.( labeled at adenine (Malanga and Althaus

  4. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650 nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20 μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy.

  5. DNA gyrase and topoisomerase IV mutations in an in vitro fluoroquinolone-resistant Coxiella burnetii strain.

    PubMed

    Vranakis, Iosif; Sandalakis, Vassilios; Chochlakis, Dimosthenis; Tselentis, Yannis; Psaroulaki, Anna

    2010-06-01

    The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a vacuole with lysosomal characteristics. Quinolones have been used as an alternative therapy for Q fever. In this study, quinolone-resistance-determining regions of the genes coding for DNA gyrase and topoisomerase IV were analyzed by DNA sequencing from an in vitro fluoroquinolone-resistant C. burnetii strain (Q212). Sequencing and aligning of DNA gyrase encoding genes (gyrA and gyrB) and topoisomerase IV genes (parC and parE) revealed one gyrA mutation leading to the amino acid substitution Asp87Gly (Escherichia coli numbering), two gyrB mutations leading to the amino acid substitutions Ser431Pro and Met518Ile, and three parC mutations leading to the amino acid substitutions Asp69Asn, Thr80Ile, and Gly104Ser. The corresponding alignment of the C. burnetii Q212 reference strain, the in vitro developed fluoroquinolone-resistant C. burnetii Q212 strain, and E. coli resulted in the identification of several other naturally occurring mutations within and outside the quinolone-resistance-determining regions of C. burnetii providing indications of possible natural resistance to fluoroquinolones. The present study adds additional potential mutations in the DNA topoisomerases that may be involved in fluoroquinolone resistance in C. burnetii due to their previous characterization in other bacterial species.

  6. A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

    PubMed Central

    Laurenceau, Raphaël; Péhau-Arnaudet, Gérard; Baconnais, Sonia; Gault, Joseph; Malosse, Christian; Dujeancourt, Annick; Campo, Nathalie; Chamot-Rooke, Julia; Le Cam, Eric; Claverys, Jean-Pierre; Fronzes, Rémi

    2013-01-01

    Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material. PMID:23825953

  7. Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification

    PubMed Central

    Seol, Yeonee; Hardin, Ashley H.; Strub, Marie-Paule; Charvin, Gilles; Neuman, Keir C.

    2013-01-01

    Type II topoisomerases are essential enzymes that regulate DNA topology through a strand-passage mechanism. Some type II topoisomerases relax supercoils, unknot and decatenate DNA to below thermodynamic equilibrium. Several models of this non-equilibrium topology simplification phenomenon have been proposed. The kinetic proofreading (KPR) model postulates that strand passage requires a DNA-bound topoisomerase to collide twice in rapid succession with a second DNA segment, implying a quadratic relationship between DNA collision frequency and relaxation rate. To test this model, we used a single-molecule assay to measure the unlinking rate as a function of DNA collision frequency for Escherichia coli topoisomerase IV (topo IV) that displays efficient non-equilibrium topology simplification activity, and for E. coli topoisomerase III (topo III), a type IA topoisomerase that unlinks and unknots DNA to equilibrium levels. Contrary to the predictions of the KPR model, topo IV and topo III unlinking rates were linearly related to the DNA collision frequency. Furthermore, topo III exhibited decatenation activity comparable with that of topo IV, supporting proposed roles for topo III in DNA segregation. This study enables us to rule out the KPR model for non-equilibrium topology simplification. More generally, we establish an experimental approach to systematically control DNA collision frequency. PMID:23460205

  8. RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

    SciTech Connect

    Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; Hura, Greg L.; Xiao, Andrew T.; Tainer, John A.; Hendzel, Michael J.; Glover, J. N. Mark

    2016-02-22

    DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

  9. RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

    DOE PAGES

    Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

    2016-02-22

    DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

  10. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

    PubMed Central

    Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

    2016-01-01

    Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. PMID:27588098

  11. A Novel Benzocoumarin-Stilbene Hybrid as a DNA ligase I inhibitor with in vitro and in vivo anti-tumor activity in breast cancer models.

    PubMed

    Hussain, Mohd Kamil; Singh, Deependra Kumar; Singh, Akhilesh; Asad, Mohd; Ansari, Mohd Imran; Shameem, Mohammad; Krishna, Shagun; Valicherla, Guru R; Makadia, Vishal; Meena, Sanjeev; Deshmukh, Amit Laxmikant; Gayen, Jiaur R; Imran Siddiqi, Mohammad; Datta, Dipak; Hajela, Kanchan; Banerjee, Dibyendu

    2017-09-06

    Existing cancer therapies are often associated with drug resistance and toxicity, which results in poor prognosis and recurrence of cancer. This necessitates the identification and development of novel therapeutics against existing as well as novel cellular targets. In this study, a novel class of Benzocoumarin-Stilbene hybrid molecules were synthesized and evaluated for their antiproliferative activity against various cancer cell lines followed by in vivo antitumor activity in a mouse model of cancer. The most promising molecule among the series, i.e. compound (E)-4-(3,5-dimethoxystyryl)-2H-benzo[h]chromen-2-one (19) showed maximum antiproliferative activity in breast cancer cell lines (MDA-MB-231 and 4T1) and decreased the tumor size in the in-vivo 4T1 cell-induced orthotopic syngeneic mouse breast cancer model. The mechanistic studies of compound 19 by various biochemical, cell biology and biophysical approaches suggest that the compound binds to and inhibits the human DNA ligase I enzyme activity that might be the cause for significant reduction in tumor growth and may constitute a promising next-generation therapy against breast cancers.

  12. Mutational analysis of bacteriophage T4 RNA ligase 1. Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction.

    PubMed

    Wang, Li Kai; Ho, C Kiong; Pei, Yi; Shuman, Stewart

    2003-08-08

    T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 N zeta on the alpha phosphorus of ATP. Mutation of Lys-99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2. Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5'-phosphorylated RNA but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway.

  13. Photolysis and thermolysis of platinum(IV) 2,2'-bipyridine complexes lead to identical platinum(II)-DNA adducts.

    PubMed

    Loup, Christophe; Tesouro Vallina, Ana; Coppel, Yannick; Létinois, Ulla; Nakabayashi, Yasuo; Meunier, Bernard; Lippert, Bernhard; Pratviel, Geneviève

    2010-10-04

    Two Pt(IV) and two Pt(II) complexes containing a 2,2'-bipyridine ligand were treated with a short DNA oligonucleotide under light irradiation at 37°C or in the dark at 37 and 50°C. Photolysis and thermolysis of the Pt(IV) complexes led to spontaneous reduction of the Pt(IV) to the corresponding Pt(II) complexes and to binding of Pt(II) 2,2'-bipyridine complexes to N7 of guanine. When the reduction product was [Pt(bpy)Cl(2)], formation of bis-oligonucleotide adducts was observed, whereas [Pt(bpy)(MeNH(2))Cl](+) gave monoadducts, with chloride ligands substituted in both cases. Neither in the dark nor under light irradiation was the reductive elimination process of these Pt(IV) complexes accompanied by oxidative DNA damage. This work raises the question of the stability of photoactivatable Pt(IV) complexes toward moderate heating conditions.

  14. Novel symmetric and asymmetric DNA scission determinants for Streptococcus pneumoniae topoisomerase IV and gyrase are clustered at the DNA breakage site.

    PubMed

    Leo, Elisabetta; Gould, Katherine A; Pan, Xiao-Su; Capranico, Giovanni; Sanderson, Mark R; Palumbo, Manlio; Fisher, L Mark

    2005-04-08

    Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/c)(A/t)A*GNNCt(T/a)N(C/a) and GN4G(G/c)(A/c)G*GNNCtTN(C/a) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.

  15. Quercetin diacylglycoside analogues showing dual inhibition of DNA gyrase and topoisomerase IV as novel antibacterial agents.

    PubMed

    Hossion, Abugafar M L; Zamami, Yoshito; Kandahary, Rafiya K; Tsuchiya, Tomofusa; Ogawa, Wakano; Iwado, Akimasa; Sasaki, Kenji

    2011-06-09

    A structure-guided molecular design approach was used to optimize quercetin diacylglycoside analogues that inhibit bacterial DNA gyrase and topoisomerase IV and show potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. In this paper, such novel 3,7-diacylquercetin, quercetin 6''-acylgalactoside, and quercetin 2'',6''-diacylgalactoside analogues of lead compound 1 were prepared to assess their target specificities and preferences in bacteria. The significant enzymatic inhibition of both Escherichia coli DNA gyrase and Staphylococcus aureus topoIV suggest that these compounds are dual inhibitors. Most of the investigated compounds exhibited pronounced inhibition with MIC values ranging from 0.13 to 128 μg/mL toward the growth of multidrug-resistant Gram-positive methicillin-resistant S. aureus, methicillin sensitive S. aureus, vancomycin-resistant enterococci (VRE), vancomycin intermediate S. aureus, and Streptococcus pneumoniae bacterial strains. Structure-activity relationship studies revealed that the acyl moiety was absolutely essential for activity against Gram-positive organisms. The most active compound 5i was 512-fold more potent than vancomycin and 16-32-fold more potent than 1 against VRE strains. It also has realistic in situ intestinal absorption in rats and showed very low acute toxicity in mice. So far, this compound can be regarded as a leading antibacterial agent.

  16. Cooperative assembly of a protein-DNA filament for nonhomologous end joining.

    PubMed

    Tsai, Chun J; Chu, Gilbert

    2013-06-21

    Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3' overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, whereas XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to five molecules each of XL and XLF into a Ku-XL-XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL, and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to six molecules each of Ku and XL into a Ku-XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular mass, MEnd ligase-DNA had a lower electrophoretic mobility than Ku-XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggests that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation.

  17. Cooperative Assembly of a Protein-DNA Filament for Nonhomologous End Joining*

    PubMed Central

    Tsai, Chun J.; Chu, Gilbert

    2013-01-01

    Nonhomologous end joining repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. Ku, XRCC4/Ligase IV (XL), and XLF have a remarkable mismatched end (MEnd) ligase activity, particularly for ends with mismatched 3′ overhangs, but the mechanism has remained obscure. Here, we showed XL required Ku to bind DNA, whereas XLF required both Ku and XL to bind DNA. We detected cooperative assembly of one or two Ku molecules and up to five molecules each of XL and XLF into a Ku-XL-XLF-DNA (MEnd ligase-DNA) complex. XLF mutations that disrupted its interactions with XRCC4 or DNA also disrupted complex assembly and end joining. Together with published co-crystal structures of truncated XRCC4 and XLF proteins, our data with full-length Ku, XL, and XLF bound to DNA indicate assembly of a filament containing Ku plus alternating XL and XLF molecules. By contrast, in the absence of XLF, we detected cooperative assembly of up to six molecules each of Ku and XL into a Ku-XL-DNA complex, consistent with a filament containing alternating Ku and XL molecules. Despite a lower molecular mass, MEnd ligase-DNA had a lower electrophoretic mobility than Ku-XL-DNA. The anomalous difference in mobility and difference in XL to Ku molar ratio suggests that MEnd ligase-DNA has a distinct structure that successfully aligns mismatched DNA ends for ligation. PMID:23620595

  18. ct-DNA Binding and Antibacterial Activity of Octahedral Titanium (IV) Heteroleptic (Benzoylacetone and Hydroxamic Acids) Complexes

    PubMed Central

    Kaushal, Raj; Thakur, Sheetal; Nehra, Kiran

    2016-01-01

    Five structurally related titanium (IV) heteroleptic complexes, [TiCl2(bzac)(L1–4)] and [TiCl3(bzac)(HL5)]; bzac = benzoylacetonate; L1–5 = benzohydroximate (L1), salicylhydroximate (L2), acetohydroximate (L3), hydroxyurea (L4), and N-benzoyl-N-phenyl hydroxylamine (L5), were used for the assessment of their antibacterial activities against ten pathogenic bacterial strains. The titanium (IV) complexes (1–5) demonstrated significant level of antibacterial properties as measured using agar well diffusion method. UV-Vis absorption spectroscopic technique was applied, to get a better insight into the nature of binding between titanium (IV) complexes with calf thymus DNA (ct-DNA). On the basis of the results of UV-Vis absorption spectroscopy, the interaction between ct-DNA and the titanium (IV) complexes is likely to occur through the same mode. Results indicated that titanium (IV) complex can bind to calf thymus DNA (ct-DNA) via an intercalative mode. The intrinsic binding constant (Kb) was calculated by absorption spectra by using Benesi-Hildebrand equation. Further, Gibbs free energy was also calculated for all the complexes. PMID:27119022

  19. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

    PubMed

    Guzmán-Herrador, Dolores L; Steiner, Samuel; Alperi, Anabel; González-Prieto, Coral; Roy, Craig R; Llosa, Matxalen

    2017-01-01

    We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

  20. Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes.

    PubMed

    Al-Hazmi, G A; El-Metwally, N M; El-Gammal, O A; El-Asmy, A A

    2008-01-01

    The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy-p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H(2)PtCl(6) produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt(oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms.

  1. Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes

    NASA Astrophysics Data System (ADS)

    Al-Hazmi, G. A.; El-Metwally, N. M.; El-Gammal, O. A.; El-Asmy, A. A.

    2008-01-01

    The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy- p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H 2PtCl 6 produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt( oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms.

  2. Dual targeting of DNA gyrase and topoisomerase IV: target interactions of heteroaryl isothiazolones in Staphylococcus aureus.

    PubMed

    Cheng, Jijun; Thanassi, Jane A; Thoma, Christy L; Bradbury, Barton J; Deshpande, Milind; Pucci, Michael J

    2007-07-01

    Heteroaryl isothiazolones (HITZs) are antibacterial agents that display excellent in vitro activity against Staphylococcus aureus. We recently identified a series of these compounds that show potent bactericidal activities against methicillin-resistant Staphylococcus aureus (MRSA). We report here the results of in vitro resistance studies that reveal potential underlying mechanisms of action. HITZs selected gyrA mutations exclusively in first-step mutants of wild-type S. aureus, indicating that in contrast to the case with most quinolones, DNA gyrase is the primary target. The compounds displayed low mutation frequencies (10(-9) to 10(-10)) at concentrations close to the MICs and maintained low MICs (< or =0.016 microg/ml) against mutants with single mutations in either gyrA or grlA (parC). These data suggested that HITZs possess significant inhibitory activities against target enzymes, DNA gyrase and topoisomerase IV. This dual-target inhibition was supported by low 50% inhibitory concentrations against topoisomerase IV as measured in a decatenation activity assay and against DNA gyrase as measured in a supercoiling activity assay. Good antibacterial activities (< or =1 microg/ml) against staphylococcal gyrA grlA double mutants, as well as low frequencies (10(-9) to 10(-10)) of selection of still higher-level mutants, also suggested that HITZs remained active against mutant enzymes. We further demonstrated that HITZs exhibit good inhibition of both S. aureus mutant enzymes and thus continue to possess a novel dual-targeting mode of action against these mutant strains. In stepwise acquisition of mutations, HITZs selected quinolone resistance determining region mutations gyrA(Ser84Leu), grlA(Ser80Phe), grlA(Ala116Val), and gyrA(Glu88Lys) sequentially, suggesting that the corresponding amino acids are key amino acids involved in the binding of HITZs to topoisomerases. The overall profile of these compounds suggests the potential utility of HITZs in combating

  3. Unique structural features in DNA polymerase IV enable efficient bypass of the N2 adduct induced by the nitrofurazone antibiotic.

    PubMed

    Kottur, Jithesh; Sharma, Amit; Gore, Kiran R; Narayanan, Naveen; Samanta, Biswajit; Pradeepkumar, Pushpangadan I; Nair, Deepak T

    2015-01-06

    The reduction in the efficacy of therapeutic antibiotics represents a global problem of increasing intensity and concern. Nitrofuran antibiotics act primarily through the formation of covalent adducts at the N(2) atom of the deoxyguanosine nucleotide in genomic DNA. These adducts inhibit replicative DNA polymerases (dPols), leading to the death of the prokaryote. N(2)-furfuryl-deoxyguanosine (fdG) represents a stable structural analog of the nitrofuran-induced adducts. Unlike other known dPols, DNA polymerase IV (PolIV) from E. coli can bypass the fdG adduct accurately with high catalytic efficiency. This property of PolIV is central to its role in reducing the sensitivity of E. coli toward nitrofuran antibiotics such as nitrofurazone (NFZ). We present the mechanism used by PolIV to bypass NFZ-induced adducts and thus improve viability of E. coli in the presence of NFZ. Our results can be used to develop specific inhibitors of PolIV that may potentiate the activity of nitrofuran antibiotics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Types of Ubiquitin Ligases.

    PubMed

    Morreale, Francesca Ester; Walden, Helen

    2016-03-24

    Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

  5. Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.

    PubMed

    Hamada, Mariko; Shimase, Koji; Noda, Keiichi; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2013-05-01

    We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

  6. Pharmacological evaluation of poly(3-methylthiophene) and its titanium(IV)phosphate nanocomposite: DNA interaction, molecular docking, and cytotoxic activity.

    PubMed

    Baig, Umair; Gondal, M A; Alam, Md Fazle; Wani, Waseem A; Younus, Hina

    2016-11-01

    Cancer and pathogenic microbial diseases have terribly affected human health over a longer period of time. In response to the increasing casualties due to cancer and microbial diseases, unique poly(3-methylthiophene) and poly(3-methylthiophene)-titanium(IV)phosphate composite were prepared via in-situ oxidative chemical polymerization in this work. The poly(3-methylthiophene) and poly(3-methylthiophene)-titanium(IV)phosphate composite were well characterized by Fourier transform infrared spectroscopy and field emission scanning electron microscopy. DNA binding studies by UV-Visible and fluorescence spectroscopic investigations indicated strong binding affinities of poly(3-methylthiophene) and poly(3-methylthiophene)-titanium(IV)phosphate nanocomposite; leading to structural damage of DNA. Poly(3-methylthiophene)-titanium(IV)phosphate nanocomposite showed stronger interactions with DNA as compared to poly(3-methylthiophene) and from dye displacement assay it was confirmed that mode of binding of both the formulations was intercalative. The antimicrobial screening revealed that polymer and its composite displayed stronger antibacterial effects than ampicillin against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhimurium. Besides, the poly(3-methylthiophene) and poly(3-methylthiophene)-titanium(IV)phosphate nanocomposite showed dose dependent effects towards estrogen receptor positive breast cancer (MCF-7) and estrogen receptor negative breast cancer (MDA-MB-231) cell lines; with poly(3-methylthiophene)-titanium(IV)phosphate nanocomposite showing better activities against both cell lines. In all in-vitro biological investigations, poly(3-methylthiophene)-titanium(IV)phosphate composite showed superior properties to that of the pure poly(3-methylthiophene), which encouraged us to suggest its potential as future therapeutic gear in drug delivery and other allied fields.

  7. Synthesis and properties of new DNA cleavage agents based on oxoruthenium(IV)

    SciTech Connect

    Gupta, N.; Grover, N.; Neyhart, G.A.; Singh, P.; Thorp, H.H. )

    1993-02-03

    New aquaruthenium(II) reagents that are capable of being oxidized to hydrooxoruthenium(III) and oxoruthenium(IV) have been prepared. Complexes based on Ru(tpy)(L)OH[sub 2][sup 2+] (L = [eta][sup 2]-tpt, phen, dppz, tmen; tpy = 2,2[prime]:6[prime],2[double prime]-terpyridine, tpt = 2,4,6-tripyridyltriazine, phen = 1,10-phenanthroline, dppz = dipyridophenazine, and tmen = N,N,N[prime],N[prime]-tetramethylethylenediamine) have been prepared and can all be reversibly oxidized to their Ru[sup IV]O forms, which are component DNA cleavage agents, as in Ru(phen)[sub 2](py)O[sup 2+]. In addition to Ru(tpy)([eta][sup 2]-tpt)OH[sub 2][sup 2+], the [eta][sup 3] complex of tpt, Ru(tpy)([eta][sup 3]-tpt)[sup 2+], can also be prepared under similar conditions. In the presence of Ag[sup +] ion, a novel Ru[sub 2]Ag complex can be isolated and has been crystallographically characterized. The complex [Ru(tpy)([eta][sup 3]-tpt)](ClO[sub 4])[sub 2][center dot]0.5AgClO[sub 4][center dot]0.5H[sub 2]O crystallizes in the monoclinic space group A2/A with a = 14.723 (5) [Angstrom], b = 26.061 (6) [Angstrom], c = 22.148 (6) [Angstrom], [beta] = 106.33 (3)[degrees], V = 8155 (5) [Angstrom][sup 3], Z = 4, R = 0.0807, and R[sub w] = 0.1156 for 2,923 reflections with I [ge] 2[sigma](I). The Ru(tpy)OH[sub 2][sup 2+] unit can also be attached to the tmen-AO[sup +] ligand, where a N,N[prime],N[prime]-trimethylethylenediamine function is appended via a (CH[sub 2])[sub 6] linker to the acridine orange intercalator. The Ru(tpy)(tmen-AO)OH[sub 2][sup 3+] complex is an effective cleavage agent, but only when oxidation is performed on the complex prebound to DNA. In homogeneous solution, electrochemically reversible access of only the Ru[sup III]OH form is possible, probably because of oxidation of the polymethylene linker. 25 refs., 11 figs., 9 tabs.

  8. Sequence studies on mouse L-cell satellite DNA by base-specific degradation with T4 endonuclease IV.

    PubMed

    Harbers, K; Spencer, J H

    1978-10-24

    The base sequence of mouse L-cell satellite DNA was investigated by degradation of the two separated complementary strands with the base specific enzyme, T4 endonuclease IV. Digestion of the heavy strand DNA released a limited number of oligonucleotides which were separated by ionophoresis/homochromatography, isolated, and sequenced by the 'wandering spot' method. The light strand DNA was resistant to digestion with T4 endonuclease IV and no detectable amounts of oligonucleotides were released. The oligonucleotides obtained from the heavy strand were related in sequence, indicating that mouse satellite DNA derived from a short tandemly repeated sequence. The sequence of part of the original repeat unit is proposed to be C-A-T-T-T-T-T-C. Five major oligonucleotides were identified, all of which differ from the proposed original sequence by single base changes. The five major oligonucleotides occur with about equal frequency and together comprise approximately 50% of the oligonucleotides released by T4 endonuclease IV from the heavy strand DNA. In addition to the five major oligonucleotides, several oligonucleotides were found to occur in lesser amounts. Since these oligonucleotides are related to the major oligonucleotides, it is likely that they have arisen from them by mutation.

  9. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks.

    PubMed

    Nagaki, S; Yamamoto, M; Yumoto, Y; Shirakawa, H; Yoshida, M; Teraoka, H

    1998-05-08

    DNA ligase IV in a complex with XRCC4 is responsible for DNA end-joining in repair of DNA double-strand breaks (DSB) and V(D)J recombination. We found that non-histone chromosomal high mobility group (HMG) proteins 1 and 2 enhanced the ligation of linearized pUC119 DNA with DNA ligase IV from rat liver nuclear extract. Intra-molecular and inter-molecular ligations of cohesive-ended and blunt-ended DNA were markedly stimulated by HMG1 and 2. Recombinant HMG2-domain A, B, and (A + B) polypeptides were similarly, but non-identically, effective for the stimulation of DSB ligation reaction. Ligation of single-strand breaks (nicks) was only slightly activated by the HMG proteins. The DNA end-binding Ku protein singly or in combination with the catalytic component of DNA-dependent protein kinase (DNA-PK) as the DNA-PK holoenzyme was ineffective for the ligation of linearized pUC119 DNA. Although the stimulatory effect of HMG1 and 2 on ligation of DSB in vitro was not specific to DNA ligase IV, these results suggest that HMG1 and 2 are involved in the final ligation step in DNA end-joining processes of DSB repair and V(D)J recombination.

  10. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

    PubMed Central

    Guzmán-Herrador, Dolores L.; Steiner, Samuel; Alperi, Anabel; González-Prieto, Coral; Roy, Craig R.; Llosa, Matxalen

    2017-01-01

    We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors. PMID:28878740

  11. A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli

    PubMed Central

    Sharma, Amit; Kottur, Jithesh; Narayanan, Naveen; Nair, Deepak T.

    2013-01-01

    The Y-family DNA polymerase IV or PolIV (Escherichia coli) is the founding member of the DinB family and is known to play an important role in stress-induced mutagenesis. We have determined four crystal structures of this enzyme in its pre-catalytic state in complex with substrate DNA presenting the four possible template nucleotides that are paired with the corresponding incoming nucleotide triphosphates. In all four structures, the Ser42 residue in the active site forms interactions with the base moieties of the incipient Watson–Crick base pair. This residue is located close to the centre of the nascent base pair towards the minor groove. In vitro and in vivo assays show that the fidelity of the PolIV enzyme increases drastically when this Ser residue was mutated to Ala. In addition, the structure of PolIV with the mismatch A:C in the active site shows that the Ser42 residue plays an important role in stabilizing dCTP in a conformation compatible with catalysis. Overall, the structural, biochemical and functional data presented here show that the Ser42 residue is present at a strategic location to stabilize mismatches in the PolIV active site, and thus facilitate the appearance of transition and transversion mutations. PMID:23525461

  12. Structural Insights into the Fluoroquinolone Resistance Mechanism of Shigella flexneri DNA Gyrase and Topoisomerase IV.

    PubMed

    Tamanna; Ramana, Jayashree

    2016-07-01

    Traveler's diarrhea (TD) is an important public health concern that can result from a variety of intestinal pathogens, including bacteria, parasites, and virus. A number of antibiotics are being used to cure TD, but due to widespread use of these antibiotics, the pathogens are becoming resistant to them. In this work, we performed docking studies of DNA gyraseA (GyrA) and topoisomerase IV (ParC) of Shigella flexneri and their mutants with two different fluoroquinolones, ciprofloxacin and norfloxacin, to understand their resistance mechanism at the structural level. S. flexneri strains with mutations at serine 83 to leucine and aspartic acid 87 to glutamate or asparagine of GyrA and that of serine 80 to isoleucine in ParC have decreased susceptibility to fluoroquinolones. This analysis revealed that interaction of ciprofloxacin/norfloxacin with all the mutants was weaker than the interaction of ciprofloxacin/norfloxacin with the wild type. This study highlights the importance of aspartic acid and serine in GyrA and that of serine in ParC, forming bonds with ciprofloxacin/norfloxacin, which may play a crucial role in antibiotic resistance. This work corelates very well with the experimental outcomes and gives a good explanation for fluoroquinolone resistance in S. flexneri.

  13. Microhomology-mediated DNA strand annealing and elongation by human DNA polymerases λ and β on normal and repetitive DNA sequences

    PubMed Central

    Crespan, Emmanuele; Czabany, Tibor; Maga, Giovanni; Hübscher, Ulrich

    2012-01-01

    ‘Classical’ non-homologous end joining (NHEJ), dependent on the Ku70/80 and the DNA ligase IV/XRCC4 complexes, is essential for the repair of DNA double-strand breaks. Eukaryotic cells possess also an alternative microhomology-mediated end-joining (MMEJ) mechanism, which is independent from Ku and DNA ligase 4/XRCC4. The components of the MMEJ machinery are still largely unknown. Family X DNA polymerases (pols) are involved in the classical NHEJ pathway. We have compared in this work, the ability of human family X DNA pols β, λ and μ, to promote the MMEJ of different model templates with terminal microhomology regions. Our results reveal that DNA pol λ and DNA ligase I are sufficient to promote efficient MMEJ repair of broken DNA ends in vitro, and this in the absence of auxiliary factors. However, DNA pol β, not λ, was more efficient in promoting MMEJ of DNA ends containing the (CAG)n triplet repeat sequence of the human Huntingtin gene, leading to triplet expansion. The checkpoint complex Rad9/Hus1/Rad1 promoted end joining by DNA pol λ on non-repetitive sequences, while it limited triplet expansion by DNA pol β. We propose a possible novel role of DNA pol β in MMEJ, promoting (CAG)n triplet repeats instability. PMID:22373917

  14. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    SciTech Connect

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P.

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves stacking of the AFB{sub 1

  15. Exploration of DNA binding mode, chemical nuclease, cytotoxic and apoptotic potentials of diketone based oxovanadium(IV) complexes.

    PubMed

    Inamdar, Poonam Rajiv; Sheela, Angappan

    2015-05-01

    Two diketone based oxovanadium complexes, viz., bis(4,4,4-trifluoro-1-phenylbutane-1,3-dionato)oxovanadium(IV) (1) and bis(1,1,1-trifluoropentane-2,4-dionato)oxovanadium(IV) (2), have been synthesized and characterized by spectroscopic and analytical techniques. The DNA binding and the cleaving ability of the complexes is assessed by UV-vis spectroscopy, fluorescence spectroscopy, viscometry and gel electrophoretic studies. The DNA binding constant values (Kb) are found to be 1.95 ± 0.16 × 10(3)M(-1) for complex 1 and 1.064 ± 0.17 × 10(3)M(-1) for complex 2, respectively. Based on the results of the spectral and viscosity studies, it is observed that the complexes, interestingly, have preferred minor groove binding with DNA. Further, the concentration-dependent oxidative cleavage pattern of pBR322 in the presence of the activating reagent, hydrogen peroxide, has also been discussed. In addition, the complexes have shown moderate cytotoxic activity by inducing apoptosis against the cervical cancer cell line, HeLa. The results of in silico analysis and logP predictions are found to be in good agreement with the experimental observations. Thus, synthesized oxovanadium complexes have displayed promising DNA binding behavior and DNA cleavage activity with moderately cytotoxic nature.

  16. Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2+ to the second metal binding site

    PubMed Central

    Oppegard, Lisa M.; Schwanz, Heidi A.; Towle, Tyrell R.; Kerns, Robert J.; Hiasa, Hiroshi

    2016-01-01

    Background Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. Methods We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2+-, Mn2+-, or Ca2+-supported DNA cleavage activity of Esherichia coli Topo IV. Results In the absence of any drug, 20–30 mM Mg2+ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn2+ or Ca2+ was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2+ concentrations where Topo IV alone could not efficiently cleave DNA. Conclusions and General Significance At low Mg2+ concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg2+ binding to metal binding site B through the structural distortion in DNA. As Mg2+ concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg2+ at site B or inhibition the binding of Mg2+ to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg2+ binding. PMID:26723176

  17. Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg(2+) to the second metal binding site.

    PubMed

    Oppegard, Lisa M; Schwanz, Heidi A; Towle, Tyrell R; Kerns, Robert J; Hiasa, Hiroshi

    2016-03-01

    Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV. In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA. At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Two divergent members of a tobacco 4-coumarate:coenzyme A ligase (4CL) gene family. cDNA structure, gene inheritance and expression, and properties of recombinant proteins.

    PubMed Central

    Lee, D; Douglas, C J

    1996-01-01

    Several cDNA clones encoding 4-coumarate:coenzyme A ligase (4CL) were isolated from a tobacco (Nicotiana tabacum) cDNA library and grouped into two classes. Sequencing of one cDNA from each class showed that the clones were similar to other 4CL genes and about 80% identical with each other. Genomic Southern blots using DNA from Nicotiana sylvestris, Nicotiana tomentosiformis, and N. tabacum demonstrated the presence of both classes of 4CL sequences (4CL1 and 4CL2) in the progenitor species and in tobacco. Northern blots indicated that 4CL mRNA transcripts are highest in old stems and higher in the unpigmented corolla tubes than in the pigmented limbs of tobacco flowers. The 4CL genes are developmentally regulated and are wound and methyl jasmonate inducible. The relative abilities of recombinant 4CL1 and 4CL2 proteins to utilize 4-coumarate, ferulate, and caffeate as substrates were similar and comparable with that of 4CL in tobacco stem extracts. Surprisingly, both recombinant 4CL proteins utilized cinnamate as a substrate, an activity not observed in stem extracts. This activity was inhibited by a heat-labile, high-molecular-weight factor found in tobacco stem extracts, suggesting that the substrate specificity of 4CL is, in part, determined by the activity of proteinaceous cellular components. PMID:8819324

  19. The conserved Fanconi anemia nuclease Fan1 and the SUMO E3 ligase Pli1 act in two novel Pso2-independent pathways of DNA interstrand crosslink repair in yeast

    PubMed Central

    Fontebasso, Y.; Etheridge, T.J.; Oliver, A.W.; Murray, J.M.; Carr, A.M.

    2013-01-01

    DNA interstrand cross-links (ICLs) represent a physical barrier to the progression of cellular machinery involved in DNA metabolism. Thus, this type of adduct represents a serious threat to genomic stability and as such, several DNA repair pathways have evolved in both higher and lower eukaryotes to identify this type of damage and restore the integrity of the genetic material. Human cells possess a specialized ICL-repair system, the Fanconi anemia (FA) pathway. Conversely yeasts rely on the concerted action of several DNA repair systems. Recent work in higher eukaryotes identified and characterized a novel conserved FA component, FAN1 (Fanconi anemia-associated nuclease 1, or FANCD2/FANCI-associated nuclease 1). In this study, we characterize Fan1 in the yeast Schizosaccharomyces pombe. Using standard genetics, we demonstrate that Fan1 is a key component of a previously unidentified ICL-resolution pathway. Using high-throughput synthetic genetic arrays, we also demonstrate the existence of a third pathway of ICL repair, dependent on the SUMO E3 ligase Pli1. Finally, using sequence-threaded homology models, we predict and validate key residues essential for Fan1 activity in ICL repair. PMID:24192486

  20. The Thermus thermophilus comEA/comEC operon is associated with DNA binding and regulation of the DNA translocator and type IV pili.

    PubMed

    Salzer, Ralf; Kern, Timo; Joos, Friederike; Averhoff, Beate

    2016-01-01

    Natural transformation systems and type IV pili are linked in many naturally competent bacteria. In the Gram-negative bacterium Thermus thermophilus, a leading model organism for studies of DNA transporters in thermophilic bacteria, seven competence proteins play a dual role in both systems, whereas two competence genes, comEA and comEC, are suggested to represent unique DNA translocator proteins. Here we show that the T. thermophilus ComEA protein binds dsDNA and is anchored in the inner membrane. comEA is co-transcribed with the flanking comEC gene, and transcription of this operon is upregulated by nutrient limitation and low temperature. To our surprise, a comEC mutant was impaired in piliation. We followed this observation and uncovered that the impaired piliation of the comEC mutant is due to a transcriptional downregulation of pilA4 and the pilN both playing a dual role in piliation and natural competence. Moreover, the comEC mutation resulted in a dramatic decrease in mRNA levels of the pseudopilin gene pilA1, which is unique for the DNA transporter. We conclude that ComEC modulates transcriptional regulation of type IV pili and DNA translocator components thereby mediating a response to extracellular parameters. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Electrochemical determination of calf thymus DNA on Zr(IV) immobilized on gold-mercaptopropionic-acid self-assembled monolayer.

    PubMed

    Shervedani, Reza Karimi; Pourbeyram, Sima

    2010-02-01

    An electrochemical biosensor, constructed by immobilization of Zr(IV) on the topside of gold-mercaptopropionic acid self-assembled monolayer (Au-MPA-Zr SAM), is developed for the sensitive quantification of calf thymus DNA (ct-DNA). The sensor is based on ionic adsorption of ct-DNA from its phosphate backbone onto the Au-MPA-Zr(IV) SAM electrode. Preparation, characterization, and application of the sensor for determination of ct-DNA are described by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Osteryoung square wave voltammetry (OSWV) in the presence of an appropriate redox reaction probe. Parameters influencing the method have been tested. A linear range calibration curve from 1.0x10(-4) to 5.0x10(-7) g mL(-1) ct-DNA with a detection limit of 9.5x10(-8) g mL(-1) and mean of relative standard deviations (R.S.D) of 2.5% for n=4 at each point was observed in the best conditions by EIS. Regeneration of the surface was carried out successfully by 5 min sonication in 0.1 M KOH solution and then 1 min incubation in 1.0x10(-3) M Zr(IV) with a good reproducibility, R.S.D=1.5% for n=4 as detected by EIS. The long-term storage stability of the electrode was also studied. 2009 Elsevier B.V. All rights reserved.

  2. Conjugation of platinum(IV) complexes with chlorambucil to overcome cisplatin resistance via a "joint action" mode toward DNA.

    PubMed

    Qin, Xiaodong; Fang, Lei; Chen, Feihong; Gou, Shaohua

    2017-09-08

    Two platinum(IV) complexes were designed and prepared by conjugation of cisplatin and oxaliplatin units with a DNA-damaging agent, chlorambucil, respectively. By taking a joint action to enhance the damage of DNA, the conjugates displayed potent antitumor activity against all the tested cancer cell lines comparable to cisplatin and oxaliplatin, and notably could overcome cisplatin resistance at certain degree. Complex 4, a hybrid of cisplatin and chlorambucil, arrested the cell cycle at the S and G2 phases, distinctive from those of cisplatin and oxaliplatin. Apoptosis studies revealed that complex 4 could induce cell apoptosis significantly in both SGC7901 and SGC7901/CDDP cells. Moreover, further investigation indicated that complex 4 suppressed the drug resistance by the improvement of the platinum uptake and the inhibition of PRAP-1 protein. These results show that the "joint action" on DNA is an effective strategy to overcome cisplatin resistance. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  3. Restoration of ligase activity in E. coli K12 lig ts7 strain by bacteriophage Mu and cloning of a DNA fragment harbouring the Mu 'lig' gene.

    PubMed Central

    Ghelardini, P; Paolozzi, L; Liebart, J C

    1980-01-01

    Restoration of ligase activity has been observed in E. coli K12 ligts7 strain lysogenic for Mu, in presence as well in absence of lysogenic immunity. This restoration consist in phenotypic reversal of temperature sensitivity of E. coli ligts7 which also regain the ability to sustain the complete growth cycle of T4 lig-phages. It is possible to put under the control of the gal operon the expression of the viral gene responsible for the restoration effect. This new gene of Mu has been named 'lig'. A 5 kb fragment responsible for the reported effects and localized between genes gam and lys of Mu genome has been cloned in pBR322. This recombinant plasmid used for transforming ligts7 strain restores in it normal behaviour for ligation of Okazaki pieces. PMID:6449688

  4. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

    PubMed Central

    Fresco, L D; Buratowski, S

    1994-01-01

    Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes. Images PMID:8022828

  5. Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates.

    PubMed

    Bansal, Sandhya; Tandon, Vibha

    2011-03-01

    DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.

  6. The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

    PubMed

    Henke, Sarah K; Cronan, John E

    2016-11-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

  7. A Photoactivatable Platinum(IV) Complex Targeting Genomic DNA and Histone Deacetylases.

    PubMed

    Kasparkova, Jana; Kostrhunova, Hana; Novakova, Olga; Křikavová, Radka; Vančo, Ján; Trávníček, Zdeněk; Brabec, Viktor

    2015-11-23

    We report toxic effects of a photoactivatable platinum(IV) complex conjugated with suberoyl-bis-hydroxamic acid in tumor cells. The conjugate exerts, after photoactivation, two functions: activity as both a platinum(II) anticancer drug and histone deacetylase (HDAC) inhibitor in cancer cells. This approach relies on the use of a Pt(IV) pro-drug, acting by two independent mechanisms of biological action in a cooperative manner, which can be selectively photoactivated to a cytotoxic species in and around a tumor, thereby increasing selectivity towards cancer cells. These results suggest that this strategy is a valuable route to design new platinum agents with higher efficacy for photodynamic anticancer chemotherapy.

  8. Discovery and Characterization of a Water-Soluble Prodrug of a Dual Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV.

    PubMed

    O'Dowd, Hardwin; Shannon, Dean E; Chandupatla, Kishan R; Dixit, Vaishali; Engtrakul, Juntyma J; Ye, Zhengqi; Jones, Steven M; O'Brien, Colleen F; Nicolau, David P; Tessier, Pamela R; Crandon, Jared L; Song, Bin; Macikenas, Dainius; Hanzelka, Brian L; Le Tiran, Arnaud; Bennani, Youssef L; Charifson, Paul S; Grillot, Anne-Laure

    2015-07-09

    Benzimidazole 1 is the lead compound resulting from an antibacterial program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With the goal of improving key drug-like properties, namely, the solubility and the formulability of 1, an effort to identify prodrugs was undertaken. This has led to the discovery of a phosphate ester prodrug 2. This prodrug is rapidly cleaved to the parent drug molecule upon both oral and intravenous administration. The prodrug achieved equivalent exposure of 1 compared to dosing the parent in multiple species. The prodrug 2 has improved aqueous solubility, simplifying both intravenous and oral formulation.

  9. Discovery and Characterization of a Water-Soluble Prodrug of a Dual Inhibitor of Bacterial DNA Gyrase and Topoisomerase IV

    PubMed Central

    2015-01-01

    Benzimidazole 1 is the lead compound resulting from an antibacterial program targeting dual inhibitors of bacterial DNA gyrase and topoisomerase IV. With the goal of improving key drug-like properties, namely, the solubility and the formulability of 1, an effort to identify prodrugs was undertaken. This has led to the discovery of a phosphate ester prodrug 2. This prodrug is rapidly cleaved to the parent drug molecule upon both oral and intravenous administration. The prodrug achieved equivalent exposure of 1 compared to dosing the parent in multiple species. The prodrug 2 has improved aqueous solubility, simplifying both intravenous and oral formulation. PMID:26191374

  10. Discovery of a novel azaindole class of antibacterial agents targeting the ATPase domains of DNA gyrase and Topoisomerase IV.

    PubMed

    Manchester, John I; Dussault, Daemian D; Rose, Jonathan A; Boriack-Sjodin, P Ann; Uria-Nickelsen, Maria; Ioannidis, Georgine; Bist, Shanta; Fleming, Paul; Hull, Kenneth G

    2012-08-01

    We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

  11. A comprehensive package for DNA sequence analysis in FORTRAN IV for the PDP-11.

    PubMed Central

    Arnold, J; Eckenrode, V K; Lemke, K; Phillips, G J; Schaeffer, S W

    1986-01-01

    A computer package written in Fortran-IV for the PDP-11 minicomputer is described. The package's novel features are: software for voice-entry of sequence data; a less memory intensive algorithm for optimal sequence alignment; and programs that fit statistical models to nucleic acid and protein sequences. PMID:3003673

  12. Zirconium(IV)-Catalyzed Ring Opening of on-DNA Epoxides in Water.

    PubMed

    Fan, Lijun; Davie, Christopher P

    2017-05-04

    DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS)4 ] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA β-amino alcohols leads to a variety of biologically interesting, nonaromatic heterocycles. Under these conditions, a library of 137 million on-DNA β-amino alcohols and their cyclization products was assembled. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Expression analysis of proteins involved in the non homologous end joining DNA repair mechanism, in the bone marrow of adult de novo myelodysplastic syndromes.

    PubMed

    Economopoulou, Panagiota; Pappa, Vassiliki; Kontsioti, Frieda; Papageorgiou, Sotirios; Foukas, Periklis; Liakata, Elisavet; Economopoulou, Christina; Vassilatou, Diamantina; Ioannidou, Eleni-Dikala; Chondropoulos, Spiridon; Tsirigotis, Panagiotis; Papageorgiou, Efstathios; Dervenoulas, John; Economopoulos, Theofanis

    2010-03-01

    Myelodysplastic syndromes (MDS) are characterized by genetic instability which is associated with abnormal DNA repair mechanisms. The most lethal type of DNA damage are double strand DNA breaks (DSBs), which are mainly repaired by Non Homologous End Joining Mechanism (NHEJ), whose core enzyme components include the Ku70/Ku80 heterodimer, DNA-PKcs, XRCC4 and DNA Ligase IV. The aim of the present study was the analysis of expression of proteins required for NHEJ in bone marrow cells of adult de novo MDS and their association with clinical characteristics and prognosis. Our analysis included 48 cases of MDS; 19 RA, 5 RARS, 19 RAEB, 3 RAEB-T, 1 CMML, 1 transformation to AML according to FAB classification. The expression of the enzymes Ku70, Ku80, XRCC4, DNA-PKcs and Ligase IV was determined by Western Blotting. The mean Ligase IV expression value was significantly lower in MDS patients compared to normal controls (0.53 vs. 0.78, p = 0.03). A negative correlation was found between karyotype risk group and Ligase IV values. (p = 0.05). Moreover, Ku70 expression levels were significantly lower in patients with a good prognosis karyotype (p = 0.04). Furthermore, a negative correlation between Ku70 expression values and Hb levels was observed (p = 0.04). Finally, a positive correlation was observed between enzyme Ku70 expression values and level of blasts (p = 0.04). Our findings suppor-t a potential role of NHEJ enzyme Ligase IV in the pathogenesis of MDS. Larger numbers of cases need to be screened in order to draw definite conclusion.

  14. Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends.

    PubMed

    Drouet, Jérôme; Frit, Philippe; Delteil, Christine; de Villartay, Jean-Pierre; Salles, Bernard; Calsou, Patrick

    2006-09-22

    Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV.XRCC4.Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs. Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK.DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.

  15. Alterations in DNA Gyrase and Topoisomerase IV in Resistant Mutants of Clostridium perfringens Found after In Vitro Treatment with Fluoroquinolones

    PubMed Central

    Rafii, Fatemeh; Park, Miseon; Novak, John S.

    2005-01-01

    To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants. PMID:15673722

  16. Alterations in DNA gyrase and topoisomerase IV in resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones.

    PubMed

    Rafii, Fatemeh; Park, Miseon; Novak, John S

    2005-02-01

    To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.

  17. Chiral manganese (IV) complexes derived from Schiff base ligands: Synthesis, characterization, in vitro cytotoxicity and DNA/BSA interaction.

    PubMed

    Li, Zhen; Niu, Meiju; Chang, Guoliang; Zhao, Changqiu

    2015-12-01

    Two new couples of chiral manganese (IV) complexes with Schiff-base ligands, Λ-[Mn(R-L(1))2]·2(CH3OH) (Λ-1) and Δ-[Mn(S-L(1))2]·2(CH3OH) (Δ-1), Λ-[Mn(R-L(2))2]·(H2O)2 (Λ-2) and Δ-[Mn(S-L(2))2]·(H2O)2 (Δ-2), {H2L(1)=(R/S)-(±)-1-[(1-hydroxymethyl-propylimino)-methyl]-naphthalen-2-ol, H2L(2)=(R/S)-(±)-1-[(1-Hydroxymethyl-2-phenyl-ethylimino)-methyl]-naphthalen-2-ol} have been synthesized, and fully characterized by elemental analyses, UV-Vis spectrum, circular dichroism spectrum, FT-IR spectrum, mass spectrum, and single crystal X-ray diffraction (SXRD). The interaction of the four chiral Mn (IV) complexes with CT-DNA and BSA were also investigated by various spectroscopic techniques (UV-visible, fluorescence spectroscopic). The results show that the Δ-complexes exhibit more efficient CT-DNA interaction with respect to the Λ-complexes. All the complexes could quench the intrinsic fluorescence of BSA by a static quenching process. In addition, the vitro cytotoxicity of these complexes toward four kinds of cancerous cell lines (A549, HeLa, HL-60, and Caco-2) was assayed by the MTT method, which exhibited to be selectively active against certain cell lines.

  18. Crystal structures of Escherichia coli topoisomerase IV ParE subunit (24 and 43 kilodaltons): a single residue dictates differences in novobiocin potency against topoisomerase IV and DNA gyrase.

    PubMed

    Bellon, Steven; Parsons, Jonathan D; Wei, Yunyi; Hayakawa, Koto; Swenson, Lora L; Charifson, Paul S; Lippke, Judith A; Aldape, Robert; Gross, Christian H

    2004-05-01

    Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-A resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-A resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 micro M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K(m) increased approximately 5-fold and the V(max) decreased approximately 30%. In contrast, the topoisomerase IV ATP K(m) decreased by a factor of 6, and the V(max) increased approximately 2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.

  19. Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

    PubMed

    Mitton-Fry, Mark J; Brickner, Steven J; Hamel, Judith C; Barham, Rose; Brennan, Lori; Casavant, Jeffrey M; Ding, Xiaoyuan; Finegan, Steven; Hardink, Joel; Hoang, Thuy; Huband, Michael D; Maloney, Meghan; Marfat, Anthony; McCurdy, Sandra P; McLeod, Dale; Subramanyam, Chakrapani; Plotkin, Michael; Reilly, Usa; Schafer, John; Stone, Gregory G; Uccello, Daniel P; Wisialowski, Todd; Yoon, Kwansik; Zaniewski, Richard; Zook, Christopher

    2017-08-01

    Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC90=0.125μg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC50 of 85.9μM and a favorable profile in the anesthetized guinea pig model. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The requirement of Artemis in double-strand break repair depends on the type of DNA damage.

    PubMed

    Kurosawa, Aya; Koyama, Hideki; Takayama, Shinichi; Miki, Kensuke; Ayusawa, Dai; Fujii, Michihiko; Iiizumi, Susumu; Adachi, Noritaka

    2008-01-01

    Artemis is a recently identified factor involved in V(D)J recombination and nonhomologous end joining (NHEJ) of DNA double-strand break (DSB) repair. Here, we performed targeted disruption of the Artemis gene (ARTEMIS) in the human pre-B cell line Nalm-6. Unexpectedly, we found that cells lacking Artemis exhibit increased sensitivity to low doses, but not high doses, of ionizing radiation. We also show that ARTEMIS-deficient cells are hypersensitive to the topoisomerase II inhibitor etoposide, but to a much lesser extent than cells lacking DNA ligase IV, a critical component of NHEJ. Unlike DNA ligase IV-deficient cells, ARTEMIS-deficient cells were not hypersensitive to ICRF-193, a topoisomerase II inhibitor that does not stabilize topoisomerase II-DNA cleavable complexes. Collectively, our results suggest that Artemis only partially participates in the NHEJ pathway to repair DSBs in human somatic cells.

  1. Ubiquitin Protein Ligase Ring2 Is Involved in S-phase Checkpoint and DNA Damage in Cells Exposed to Benzo[a]pyrene.

    PubMed

    Yang, Jin; Chen, Wentao; Fan, Yanfeng; Zhang, Huitao; Wang, Wubin; Zhang, Hongjie

    2016-10-01

    Previous studies in our laboratory demonstrated that Ring2 may affect DNA damage and repair through pathways other than through regulating the expression of the nucleotide excision repair protein. In a series of experiments using wild-type cell (16HBE and WI38) and small interfering RNA (siRNA) Ring2 cells exposed to benzo[a]pyrene (BaP), we evaluated the cell cycle and DNA damage. The benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-DNA) adduct assay demonstrated that in vitro exposure to BaP increased DNA damage in a time- and dose-dependent manner in wild-type and siRNA Ring2 cells. Analysis of covariance showed that a decrease of Ring2 caused DNA hypersensitivity to BaP. Flow cytometry results and proliferating cell nuclear antigen levels indicated that inhibition of Ring2 attenuated the effect of BaP on S-phase arrest. Taken together, these data implied that the lower proportion of cells in the S phase induced by inhibition of Ring2 may play an important role in DNA hypersensitivity to BaP.

  2. Diazido mixed-amine platinum(IV) anticancer complexes activatable by visible-light form novel DNA adducts.

    PubMed

    Zhao, Yao; Woods, Julie A; Farrer, Nicola J; Robinson, Kim S; Pracharova, Jitka; Kasparkova, Jana; Novakova, Olga; Li, Huilin; Salassa, Luca; Pizarro, Ana M; Clarkson, Guy J; Song, Lijiang; Brabec, Viktor; Sadler, Peter J

    2013-07-15

    Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side-effects. Photoactivatable Pt(IV) prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X-ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (1; MA=methylamine, Py=pyridine) and trans,trans,trans-[Pt(N3)2(OH)2(MA)(Tz)] (2; Tz=thiazole), and interpret their photophysical properties by TD-DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1p and 1q. Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin-resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf-thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono- and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross-links, with evidence for DNA strand cross-linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin-type lesions. The photo-induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt(II) compounds trans-[PtCl2(MA)(Py)] (5) and trans-[PtCl2(MA)(Tz)] (6). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1, whereas the dark reactions of 5 and 6 with DNA were comparatively slow

  3. Diazido Mixed-Amine Platinum(IV) Anticancer Complexes Activatable by Visible-Light Form Novel DNA Adducts

    PubMed Central

    Zhao, Yao; Woods, Julie A; Farrer, Nicola J; Robinson, Kim S; Pracharova, Jitka; Kasparkova, Jana; Novakova, Olga; Li, Huilin; Salassa, Luca; Pizarro, Ana M; Clarkson, Guy J; Song, Lijiang; Brabec, Viktor; Sadler, Peter J

    2013-01-01

    Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side-effects. Photoactivatable PtIV prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X-ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (1; MA=methylamine, Py=pyridine) and trans,trans,trans-[Pt(N3)2(OH)2(MA)(Tz)] (2; Tz=thiazole), and interpret their photophysical properties by TD-DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1 p and 1 q. Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin-resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf-thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono- and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross-links, with evidence for DNA strand cross-linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin-type lesions. The photo-induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the PtII compounds trans-[PtCl2(MA)(Py)] (5) and trans-[PtCl2(MA)(Tz)] (6). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1, whereas the dark reactions of 5 and 6 with DNA were comparatively slow

  4. Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

    PubMed Central

    Lovett, S. T.; Sutera-Jr., V. A.

    1995-01-01

    The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),'' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517(am) as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517(am) derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor ``srjD'' is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ. PMID:7635292

  5. Emergence of fluoroquinolone-resistant Propionibacterium acnes caused by amino acid substitutions of DNA gyrase but not DNA topoisomerase IV.

    PubMed

    Nakase, Keisuke; Sakuma, Yui; Nakaminami, Hidemasa; Noguchi, Norihisa

    2016-12-01

    With the aim of elucidating the mechanisms of fluoroquinolones resistance in Propionibacterium acnes, we determined the susceptibility of fluoroquinolones in 211 isolates from patients with acne vulgaris. We identified five isolates (2.4%) with reduced susceptibility to nadifloxacin (minimum inhibitory concentration ≥ 4 μg/ml). Determination of the sequences of the DNA gyrase (gyrA and gyrB) and DNA topoisomerase (parC and parE) genes showed the amino acid substitutions Ser101Leu and Asp105Gly of GyrA in four and one of the isolates, respectively. In vitro mutation experiments showed that low-level fluoroquinolone-resistant mutants with the Ser101Leu or Asp105Gly substitution in GyrA could be obtained from selection with ciprofloxacin and levofloxacin. The pattern of substitution (Ser101Trp in GyrA) caused by nadifloxacin selection was different from that induced by the other fluoroquinolones. In the isolation of further high-level resistant mutants, acquisition of another amino acid substitution of GyrB in addition to those of GyrA was detected, but there were no substitutions of ParC and ParE. In addition, the mutant prevention concentration and mutation frequency of nadifloxacin were lowest among the tested fluoroquinolones. The growth of the Ser101Trp mutant was lower than that of the other mutants. Our findings suggest that the Ser101Trp mutant of P. acnes emerges rarely and disappears immediately, and the risk for the prevalence of fluoroquinolones-resistant P. acnes differs according to the GyrA mutation type. To our knowledge, this study is the first to demonstrate the mechanisms of resistance to fluoroquinolones in P. acnes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Accumulation of linear mitochondrial DNA fragments in the nucleus shortens the chronological life span of yeast.

    PubMed

    Cheng, Xin; Ivessa, Andreas S

    2012-10-01

    Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases. Copyright © 2012 Elsevier GmbH. All rights reserved.

  7. DNA binding, antitumor activities, and hydroxyl radical scavenging properties of novel oxovanadium (IV) complexes with substituted isoniazid.

    PubMed

    Liao, Xiangwen; Lu, Jiazheng; Ying, Peng; Zhao, Ping; Bai, Yinliang; Li, Wengjie; Liu, Mingpei

    2013-12-01

    Four novel oxovanadium(IV) complexes—[VO(PAHN)(phen)] (1; PAHN is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-naphthalenylene] hydrazide, phen is 1,10-phenanthroline), [VO(PAHN)(bpy)] (2; bpy is 2,2′-bipyridine), [VO(PAH)(phen)] (3; PAH is 4-pyridinecarboxylic acid, 2-[(2-hydroxy)-1-phenyl]methylene hydrazide), and [VO(PAH)(bpy)] (4)—have been synthesized and characterized by elemental analysis, UV–vis spectroscopy, electrospray ionization mass spectrometry, IR spectroscopy, 1H-NMR spectroscopy, and 13C-NMR spectroscopy. Their interactions with calf thymus DNA were investigated. The results suggest that these complexes bind to DNA in an intercalative mode. All four complexes exhibited highly cytotoxic activity against tumor cells (SH-SY5Y, MCF-7, and SK-N-SH), with 50 % inhibitory concentrations of the same order of magnitude as for cisplatin or of lower order of magnitude. Complex 1 exhibited the highest interaction ability and was found to be the most potent antitumor agent among the four complexes. It can cause G2/M phase arrest of the cell cycle, induces significant apoptosis in SK-N-SH cells, and displays typical morphological apoptotic characteristics. In addition, their hydroxyl radical scavenging properties have been tested, and complex 1 was the best inhibitor.

  8. Synthesis, characterization, cytotoxicity, and DNA binding of some new platinum(II) and platinum(IV) complexes with benzimidazole ligands.

    PubMed

    Utku, Semra; Gumus, Fatma; Tezcan, Seda; Serin, Mehmet Sami; Ozkul, Aykut

    2010-08-01

    In this study, two Pt(II) and three Pt(IV) complexes with the structures of [PtL(2)Cl(2)] (1), [PtL(2)I(2)] (2), [PtL(2)Cl(2)(OH)(2)] (3), [PtL(2)Cl(2)(OCOCH(3))(2)] (4), and [PtL(2)Cl(4)] (5) (L = benzimidazole as carrier ligand) were synthesized and evaluated for their in vitro antiproliferative activities against the human MCF-7, HeLa, and HEp-2 cancer cell lines. The influence of compounds 1-5 on the tertiary structure of DNA was determined by their ability to modify the electrophoretic mobility of the form I and II bands of pBR322 plasmid DNA. The inhibition of BamH1 restriction enzyme activity of compounds 1-5 was also determined. In general, it was found that compounds 1-5 were less active than cisplatin and carboplatin against MCF-7 and HeLa cell lines (except for 1, which was found to be more active than carboplatin against the MCF-7 cell line). Compounds 1 and 3 were found to be significantly more active than cisplatin and carboplatin against the HEp-2 cell line.

  9. MutS regulates access of the error-prone DNA polymerase Pol IV to replication sites: a novel mechanism for maintaining replication fidelity

    PubMed Central

    Margara, Lucía M.; Fernández, Marisa M.; Malchiodi, Emilio L.; Argaraña, Carlos E.; Monti, Mariela R.

    2016-01-01

    Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the β clamp processivity factor by competing for binding to the ring. Moreover, the MutS–β clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity. PMID:27257069

  10. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    SciTech Connect

    Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  11. Sitafloxacin: antimicrobial activity against ciprofloxacin-selected laboratory mutants of Mycoplasma genitalium and inhibitory activity against its DNA gyrase and topoisomerase IV.

    PubMed

    Deguchi, Takashi; Kikuchi, Mina; Yasuda, Mitsuru; Ito, Shin

    2015-01-01

    A sitafloxacin regimen is highly effective on Mycoplasma genitalium infections, including those caused by the mycoplasmas harboring mutant topoisomerase IV with a quinolone resistance-associated amino acid change in ParC. In this study, we evaluated sitafloxacin antimicrobial activities against M. genitalium, including the mycoplasmas with decreased susceptibilities to quinolones, by determining minimum inhibitory concentrations (MICs) for the strain ATCC 33530 and its 3 ciprofloxacin-selected mutants, for which ciprofloxacin MICs were 8-16 times higher than that for their parent strain. We also evaluated inhibitory activities against the target enzymes of M. genitalium by determining concentrations required to inhibit 50% (IC50) of the supercoiling activity of the recombinant wild-type DNA gyrase and the decatenating activities of the recombinant wild-type topoisomerase IV and the 2 types of mutant topoisomerase IV with a single amino acid change in ParC. Sitafloxacin MICs were 0.125 for the parent strain and 0.125-0.25 μg/ml for the mutants. Sitafloxacin IC50s were 3.12 for the supercoiling activity of the wild-type DNA gyrase and 2.98 μg/ml for the decatenating activity of the wild-type topoisomerase IV. Its IC50s for the decatenating activity of the mutant topoisomerase IV harboring an amino acid change in ParC were 15.1 for Gly-81 → Cys and 7.92 μg/ml for Asp-87 → Tyr. Sitafloxacin was highly active against ciprofloxacin-selected mutants of M. genitalium and possessed intense inhibitory activities not only against wild-type DNA gyrase and topoisomerase IV but also against mutant topoisomerase IV containing ParC with a quinolone resistance-associated amino acid change. Sitafloxacin could be a promising agent for M. genitalium infections. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  12. Ligase detection reaction/hybridization assays using three-dimensional microfluidic networks for the detection of low-abundant DNA point mutations.

    PubMed

    Hashimoto, Masahiko; Hupert, Mateusz L; Murphy, Michael C; Soper, Steven A; Cheng, Yu-Wei; Barany, Francis

    2005-05-15

    We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min

  13. Biological profiling of novel tricyclic inhibitors of bacterial DNA gyrase and topoisomerase IV.

    PubMed

    Savage, Victoria J; Charrier, Cédric; Salisbury, Anne-Marie; Moyo, Emmanuel; Forward, Henry; Chaffer-Malam, Nathan; Metzger, Richard; Huxley, Anthony; Kirk, Ralph; Uosis-Martin, Mario; Noonan, Gary; Mohmed, Sarfraz; Best, Stuart A; Ratcliffe, Andrew J; Stokes, Neil R

    2016-07-01

    The objective of this study was to characterize the in vitro and in vivo biological properties of a novel series of small-molecule bacterial type IIA topoisomerase inhibitors. Bacterial susceptibility testing was performed by broth microdilution. Resistance frequencies were determined by plating bacteria onto agar containing test compound and enumerating mutants. Bacteria were passaged using subinhibitory concentrations of antibacterials to generate resistance. Target enzyme inhibition was determined by exposure to antibacterials and DNA; topoisomers were visualized by gel electrophoresis. Oral and intravenous pharmacokinetic profiles were determined in mice. In vivo efficacy was determined using a mouse model of septicaemia and thigh infection with MSSA and MRSA, respectively. Representative compounds REDX04139, REDX05604 and REDX05931 demonstrated in vitro potency against a range of Gram-positive and fastidious Gram-negative pathogens. Clinical isolate testing revealed REDX04139 and REDX05931 had MIC90 values of 0.25 and 0.5 mg/L, respectively, for MRSA and MIC90 values of 2 mg/L for streptococci. REDX04139 was bactericidal in vitro against Staphylococcus aureus at 8× MIC over 6 h. Pharmacokinetic profiling of REDX04139 and REDX05604 in mice revealed low clearance and excellent bioavailability (≥71%). REDX04139 provided 100% survival against S. aureus in a mouse septicaemia model, while REDX05604 reduced bacterial load by up to 3.7 log units in the MRSA mouse thigh infection model. Redx Pharma has discovered a novel series of topoisomerase inhibitors that are being further developed for drug-resistant bacteria. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. SCF-FBXO31 E3 Ligase Targets DNA Replication Factor Cdt1 for Proteolysis in the G2 Phase of Cell Cycle to Prevent Re-replication*

    PubMed Central

    Johansson, Pegah; Jeffery, Jessie; Al-Ejeh, Fares; Schulz, Renèe B.; Callen, David F.; Kumar, Raman; Khanna, Kum Kum

    2014-01-01

    FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor. PMID:24828503

  15. Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer

    PubMed Central

    Nacerddine, Karim; Beaudry, Jean-Bernard; Ginjala, Vasudeva; Westerman, Bart; Mattiroli, Francesca; Song, Ji-Ying; van der Poel, Henk; Ponz, Olga Balagué; Pritchard, Colin; Cornelissen-Steijger, Paulien; Zevenhoven, John; Tanger, Ellen; Sixma, Titia K.; Ganesan, Shridar; van Lohuizen, Maarten

    2012-01-01

    Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis. PMID:22505453

  16. Sensitive and specific miRNA detection method using SplintR Ligase.

    PubMed

    Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M; Posfai, Janos; McReynolds, Larry A

    2016-07-27

    We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Sensitive and specific miRNA detection method using SplintR Ligase

    PubMed Central

    Jin, Jingmin; Vaud, Sophie; Zhelkovsky, Alexander M.; Posfai, Janos; McReynolds, Larry A.

    2016-01-01

    We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR® Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4–6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection. PMID:27154271

  18. Circulating Tumor DNA in Predicting Outcomes in Patients With Stage IV Head and Neck Cancer or Stage III-IV Non-small Cell Lung Cancer

    ClinicalTrials.gov

    2017-08-18

    Metastatic Squamous Neck Cancer With Occult Primary Squamous Cell Carcinoma; Salivary Gland Squamous Cell Carcinoma; Stage IIIA Non-small Cell Lung Cancer; Stage IIIB Non-small Cell Lung Cancer; Stage IV Non-small Cell Lung Cancer; Stage IV Squamous Cell Carcinoma of the Hypopharynx; Stage IV Squamous Cell Carcinoma of the Nasopharynx; Stage IVA Salivary Gland Cancer; Stage IVA Squamous Cell Carcinoma of the Larynx; Stage IVA Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage IVA Squamous Cell Carcinoma of the Oropharynx; Stage IVA Squamous Cell Carcinoma of the Paranasal Sinus and Nasal Cavity; Stage IVA Verrucous Carcinoma of the Larynx; Stage IVA Verrucous Carcinoma of the Oral Cavity; Stage IVB Salivary Gland Cancer; Stage IVB Squamous Cell Carcinoma of the Larynx; Stage IVB Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage IVB Squamous Cell Carcinoma of the Oropharynx; Stage IVB Squamous Cell Carcinoma of the Paranasal Sinus and Nasal Cavity; Stage IVB Verrucous Carcinoma of the Larynx; Stage IVB Verrucous Carcinoma of the Oral Cavity; Stage IVC Salivary Gland Cancer; Stage IVC Squamous Cell Carcinoma of the Larynx; Stage IVC Squamous Cell Carcinoma of the Lip and Oral Cavity; Stage IVC Squamous Cell Carcinoma of the Oropharynx; Stage IVC Squamous Cell Carcinoma of the Paranasal Sinus and Nasal Cavity; Stage IVC Verrucous Carcinoma of the Larynx; Stage IVC Verrucous Carcinoma of the Oral Cavity; Tongue Cancer; Untreated Metastatic Squamous Neck Cancer With Occult Primary

  19. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions.

    PubMed

    Tadi, Satish Kumar; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K; Choudhary, Bibha; Raghavan, Sathees C

    2016-01-15

    Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

  20. Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

    PubMed Central

    Tadi, Satish Kumar; Sebastian, Robin; Dahal, Sumedha; Babu, Ravi K.; Choudhary, Bibha; Raghavan, Sathees C.

    2016-01-01

    Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders. PMID:26609070

  1. Molecular Biology of STLV-III and HTLV-IV

    DTIC Science & Technology

    1990-08-22

    gel bands, pooled, and sedimented. The DNA pellet was resuspended in a lOp1 reaction volume containing T4 DNA ligase . Ligations were incubated...this procedure, all enzymes were from New England Biolabs, approximately 5u. of each restriction enzyme and approximately 3 Weiss u. of T4 DNA ligase were...volume 2-propanol. The fragments were re-ligated using T4 DNA ligase , in a 200pl reaction volume containing 15% PEG-8000, for 0.5-2hr at room temperature

  2. SCF ubiquitin ligase targeted therapies

    PubMed Central

    Skaar, Jeffrey R.; Pagan, Julia K.; Pagano, Michele

    2015-01-01

    Summary The recent clinical successes of inhibitors of the proteasome for the treatment of cancer have highlighted the therapeutic potential of this protein degradation system. Proteasome inhibitors prevent the degradation of numerous proteins, so increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a large number of processes at the cellular and organismal levels, and their misregulation is implicated in many pathologies. SCF ligases are characterized by a high specificity for their substrates, so they represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This review will explore and discuss potential strategies to target SCF-mediated biology to treat human diseases. PMID:25394868

  3. Antimicrobial susceptibility pattern and sequence analysis of DNA gyrase and DNA topoisomerase IV in Salmonella enterica serovars Typhi and Paratyphi A isolates with decreased susceptibility to ciprofloxacin.

    PubMed

    Misra, Richa; Thakare, Ritesh; Amrin, N; Prasad, Kashi Nath; Chopra, Sidharth; Dhole, Tapan Nirodhechand

    2016-08-01

    We describe the antimicrobial susceptibility pattern of 100 typhoidal Salmonella isolates recovered from blood cultures and also investigate the association of decreased ciprofloxacin susceptibility with mutations in the genes coding for DNA gyrase and topoisomerase IV in 55 isolates. The study was conducted between January 2013 and December 2015 at a tertiary care centre in north India. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion and E-test. Genotypic characterization included the screening of mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE by PCR. DNA sequence analysis was done for 55 isolates. Out of 100 isolates recovered 80 were S. Typhi, 18 were Paratyphi A and two were Paratyphi B. Eighty two percent (66/80) of S. Typhi and 15/18 S. Paratyphi A showed decreased ciprofloxacin susceptibility. The most common mutation in gyrA led to a change at codon 83 of serine to phenylalanine (n=37) or tyrosine (n=12). Five S. Typhi isolates that were resistant to ciprofloxacin (MICs of 12, 16, 24 and 32 μg/ml) had a second mutation at codon 87 in the gyrA gene changing aspartate to asparagine. There is a need to urgently review the use of fluoroquinolones for the management of enteric fever in endemic areas. © The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Molecular cloning and characterization of a putative cDNA encoding endoglucanase IV from Trichoderma viride and its expression in Bombyx mori.

    PubMed

    Li, Xing-Hua; Zhang, Peng; Liang, Shuang; Zhou, Fang; Wang, Mei-Xian; Bhaskar, Roy; Malik, Firdose Ahmad; Niu, Yan-Shan; Miao, Yun-Gen

    2012-01-01

    The development of cellulase production technology has greatly contributed to the successful use of cellulosic materials as renewable carbon sources. In this study, a putative endoglucanase IV (EG IV) complementary DNA was cloned from the mycelium of a strain of the filamentous fungus Trichoderma viride using a PCR-based exon-splicing method and expressed in both a silkworm BmN cell line and in silkworm larvae. Western blot analysis detected a band of 42 kDa in BmN cells after infection with a recombinant mBacmid/BmNPV/EG IV baculovirus. Sequence alignment analysis of the T. viride EG IV gene showed two domains that were highly conserved with glycosyl hydrolases and a funga-type cellulose-binding domain. Analysis of variance showed that silkworms infected with recombinant baculoviruses exhibited significantly higher enzyme activity that was 48.84% higher than silkworms infected with blank baculoviruses and 46.61% higher than normal silkworms. The expressed bioactive EG IV was also stable at the pH range from 5.0 to 10.0. The availability of large quantities of bioactive EG IV in silkworm provided a possibility to produce cellulase transgenic silkworm, which express bioactive cellulase specially in its digestive tract and improve its metabolism efficiency of mulberry leaves. Its application in the sericulture industry may be very promising.

  5. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus.

    PubMed

    Carr, Stephen B; Makris, George; Phillips, Simon E V; Thomas, Christopher D

    2006-11-01

    DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2(1), diffract to a resolution of 2.9 A and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 A, beta = 90.1 degrees, while crystals of GrlA59 belong to space group P2(1)2(1)2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 A. These crystals diffract to a resolution of 2.8 A. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  6. The effect of twisting motion dependent diagonal hopping on the I-V characteristics of poly(dG)-poly(dC) DNA molecule

    NASA Astrophysics Data System (ADS)

    Yudiarsah, Efta

    2017-04-01

    The effect of twisting motion dependent diagonal hopping on the I-V characteristic of Poly(dG)-Poly(dC) DNA molecule has been studied. Twisting angle dependent hopping constant is model using semi-empirical Slater-Koster theory. The I-V characteristic of DNA molecule is calculated from transmission probability using Landauer-Buttiker formalism by assuming symmetric voltage drop at the contacts. The transmission probability of charge on the molecule is calculated using transfer and scattering matrix methods, simultaneously. The calculation is carried out on 32 base pairs doubled-stranded DNA molecule model sandwiched in between two metallic electrodes. The results show that at low voltage the magnitude of current is not influenced much by the change of diagonal hopping term caused by base pair twisting motion. Larger influenced is observed at higher voltages, after decreases by increasing the coupling parameter of vibration and diagonal hopping constant up to some value, current increases. This trend is observed in the I-V characteristic of DNA molecule for all frequencies and temperatures used. Lower twisting motion frequency shows larger influences of diagonal hopping term on the current than the one at higher frequencies.

  7. Role of deoxyribonucleic acid ligase in a doxyribonucleic acid membrane fraction extracted from pneumococci.

    PubMed Central

    Greene, M; Firshein, W

    1976-01-01

    Deoxyribonucleic acid (DNA) ligase has been detected in a DNA membrane fraction extracted from Pneumococcus. The specific activity of the enzyme in this fraction is 10-fold greater than in the remaining cell extract. It remains firmly bound (with other enzymes) to the complex after a purification procedure in which a considerable percentage of the macromolecules are dissociated. The ligase acts in two ways in the DNA membrane fraction in vitro. One, it catalyzes the linkage of small-molecular-weight pieces of newly synthesized DNA into heavier-molecular-weight DNA strands as shown by others (M Gellert, 1976; R. Okazaki, A. Sugino, S. Hirose, T. Okazaki, Y. Imae, R. Kainuma-Kuroda, T. Ogawa, M. Arisawa, and Y. Kurosowa, 1973; B. Olivera and I. Lehman, 14; and A. Sugino, S. Hirose, and R. Okazaki, 1972) and, two, it protects DNA from degradation by deoxyribonucleases. This latter effect is due to a competition between the ability of the nucleases to degrade DNA and the ability of DNA ligase to seal the nicks produced by these degradative enzymes. The ligase acts cooperatively with other enzymes in the DNA membrane fraction to synthesize DNA. PMID:4433

  8. Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency.

    PubMed

    Chang, Howard H Y; Watanabe, Go; Gerodimos, Christina A; Ochi, Takashi; Blundell, Tom L; Jackson, Stephen P; Lieber, Michael R

    2016-11-18

    The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency*

    PubMed Central

    Chang, Howard H. Y.; Watanabe, Go; Gerodimos, Christina A.; Ochi, Takashi; Blundell, Tom L.; Jackson, Stephen P.; Lieber, Michael R.

    2016-01-01

    The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed. PMID:27703001

  10. DNA vaccine encoding type IV pilin of Actinobacillus pleuropneumoniae induces strong immune response but confers limited protective efficacy against serotype 2 challenge.

    PubMed

    Lu, Yu-Chun; Li, Min-Chen; Chen, Yi-Min; Chu, Chun-Yen; Lin, Shuen-Fuh; Yang, Wen-Jen

    2011-10-13

    Actinobacillus pleuropneumoniae is a gram-negative bacterial pathogen that causes swine pleuropneumonia, a highly contagious and often fatal disease that occurs worldwide. Our previous study showed that DNA vaccines encoding Apx exotoxin structural proteins ApxIA and/or ApxIIA, are a promising novel approach for immunization against the lethal challenge of A. pleuropneumoniae serotype 1. Vaccination against A. pleuropneumoniae is impeded by the lack of vaccines inducing reliable cross-serotype protection. Type IV fimbrial protein ApfA has been shown to be present and highly conserved in various serotypes of A. pleuropneumoniae. A novel DNA vaccine encoding ApfA (pcDNA-apfA) was constructed to evaluate the protective efficacy against infection with A. pleuropneumoniae serotype 2. A significant antibody response against pilin was generated following pcDNA-apfA immunization, suggesting that it was expressed in vivo. The IgG subclass (IgG1 and IgG2a) analysis indicates that the pcDNA-apfA vaccine induces both Th1 and Th2 immune responses. The IgA analysis shows that mucosal immunity could be enhanced by this DNA vaccine. Nevertheless, the strong antibody response induced by pcDNA-apfA vaccine only provided limited 30% protective efficacy against the serotype 2 challenge. These results in this study do not coincide with that the utility of type IV pilin is a good vaccine candidate against other infectious pathogens. It indicates that pilin should play a limited role in the development of a vaccine against A. pleuropneumoniae infection.

  11. Helix–hairpin–helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues

    PubMed Central

    Schomacher, Lars; Smolorz, Sabine; Ciirdaeva, Elena; Ber, Svetlana; Kramer, Wilfried; Fritz, Hans-Joachim

    2010-01-01

    The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Δung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard. PMID:20410075

  12. Endonuclease IV and exonuclease III are involved in the repair and mutagenesis of DNA lesions induced by UVB in Escherichia coli.

    PubMed

    Souza, L L; Eduardo, I R; Pádula, M; Leitão, A C

    2006-03-01

    Exonuclease III (Exo III) and endonuclease IV (Endo IV) play a critical role in the base excision repair (BER) of Escherichia coli. Both are endowed with AP endonucleolytic activity, cleaving the 5' phosphodiester bond adjacent to spontaneous or induced abasic sites in DNA. Although mutants defective in Exo III (xthA) are usually hypersensitive to oxidative agents such as hydrogen peroxide, near-UV-light and X-rays, mutants defective in Endo IV (nfo) are not as sensitive as the xthA strain. To further investigate the roles of these AP endonucleases in DNA repair, we evaluated the sensitivity and mutagenesis of xthA and nfo strains after UVB and compared with UVC light. Our results revealed that xthA but not nfo strain was hypersensitive to UVB. The use of Fe(+2) ion chelator (dipyridyl), prior to irradiation, completely protected the xthA mutant against UVB lethal lesions, suggesting the generation of toxic oxidative lesions mediated by transition metal reactions. The nfo strain displayed increased UVB-induced mutagenesis, which was significantly suppressed by pre-treatment with dipyridyl. Although xthA strain did not display increased mutagenesis after UVC and UVB treatments, this phenotype was not related to xthA mutation, but rather to an unknown secondary mutation specifying an antimutator phenotype. After UVB irradiation, the base substitution spectra of nfo strain revealed a bias towards AT-->GC transitions and GC-->CG transversions, which were also suppressed by previous treatment with the iron chelator. Overall, on the basis of the differential sensitivities and mutational spectra displayed after UVC and UVB treatments, we propose a role for Endo IV and Exo III to counteract DNA damage induced by the oxidative counterpart of UVB in E.coli.

  13. Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

    PubMed Central

    Schröder, Gunnar; Schuelein, Ralf; Quebatte, Maxime; Dehio, Christoph

    2011-01-01

    Bacterial type IV secretion systems (T4SS) mediate interbacterial conjugative DNA transfer and transkingdom protein transfer into eukaryotic host cells in bacterial pathogenesis. The sole bacterium known to naturally transfer DNA into eukaryotic host cells via a T4SS is the plant pathogen Agrobacterium tumefaciens. Here we demonstrate T4SS-mediated DNA transfer from a human bacterial pathogen into human cells. We show that the zoonotic pathogen Bartonella henselae can transfer a cryptic plasmid occurring in the bartonellae into the human endothelial cell line EA.hy926 via its T4SS VirB/VirD4. DNA transfer into EA.hy926 cells was demonstrated by using a reporter derivative of this Bartonella-specific mobilizable plasmid generated by insertion of a eukaryotic egfp-expression cassette. Fusion of the C-terminal secretion signal of the endogenous VirB/VirD4 protein substrate BepD with the plasmid-encoded DNA-transport protein Mob resulted in a 100-fold increased DNA transfer rate. Expression of the delivered egfp gene in EA.hy926 cells required cell division, suggesting that nuclear envelope breakdown may facilitate passive entry of the transferred ssDNA into the nucleus as prerequisite for complementary strand synthesis and transcription of the egfp gene. Addition of an eukaryotic neomycin phosphotransferase expression cassette to the reporter plasmid facilitated selection of stable transgenic EA.hy926 cell lines that display chromosomal integration of the transferred plasmid DNA. Our data suggest that T4SS-dependent DNA transfer into host cells may occur naturally during human infection with Bartonella and that these chronically infecting pathogens have potential for the engineering of in vivo gene-delivery vectors with applications in DNA vaccination and therapeutic gene therapy. PMID:21844337

  14. New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

    NASA Astrophysics Data System (ADS)

    Tabassum, Sartaj; Sharma, Girish Chandra; Arjmand, Farukh

    2012-05-01

    A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

  15. Accumulation of mutations in DNA gyrase and topoisomerase IV genes contributes to fluoroquinolone resistance in Vibrio cholerae O139 strains.

    PubMed

    Zhou, Yanyan; Yu, Li; Li, Jie; Zhang, Lijuan; Tong, Ying; Kan, Biao

    2013-07-01

    High resistance rates to nalidixic acid (NAL) in Vibrio cholerae serogroup O139 strains have been found, and ciprofloxacin (CIP) resistance is also observed. In this study, mutations within the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV from NAL-resistant O139 strains were analysed. The predominant mutation profile was S83I in GyrA in combination with S85L in ParC. In addition, the combination substitutions of D87N in GyrA and D420N in ParE in combination with S83I in GyrA and S85L in ParC as well as D87N in GyrA and P439S in ParE in combination with S83I in GyrA and S85L in ParC were found in the CIP-resistant strains. A series of site-directed mutants comprising D87 in GyrA, D420 in ParE and P439 in ParE were constructed from a wild-type V. cholerae O139 strain carrying the common mutations S83I in GyrA and S85L in ParC. Introduction of the mutation D87N in GyrA increased the CIP minimum inhibitory concentration (MIC) of the mutant strain by nearly 4-fold compared with the initial strain. The second introduction of D420N in ParE further significantly increased the CIP MIC to ca. 23-fold compared with the initial strain. A second introduction of P439S in ParE also increased the CIP MIC by 17-fold. Therefore, it is concluded that the emergence of D87N in GyrA and D420N or P439S in ParE dramatically induces resistance to fluoroquinolones in V. cholerae O139, and the accumulation of multiple mutations in the QRDRs confers significant resistance to fluoroquinolones in V. cholerae. Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  16. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones.

    PubMed

    Laponogov, Ivan; Pan, Xiao-Su; Veselkov, Dennis A; Cirz, Ryan T; Wagman, Allan; Moser, Heinz E; Fisher, L Mark; Sanderson, Mark R

    2016-09-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.

  17. ATM kinase activity modulates ITCH E3-ubiquitin ligase activity

    PubMed Central

    Santini, Simonetta; Stagni, Venturina; Giambruno, Roberto; Fianco, Giulia; Di Benedetto, Anna; Mottolese, Marcella; Pellegrini, Manuela; Barilà, Daniela

    2014-01-01

    Ataxia Telangiectasia Mutated (ATM) kinase, a central regulator of the DNA damage response regulates the activity of several E3-ubiquitin ligases and the ubiquitination-proteasome system is a consistent target of ATM. ITCH is an E3-ubiquitin ligase that modulates the ubiquitination of several targets, therefore participating to the regulation of several cellular responses, among which the DNA damage response, TNFα, Notch and Hedgehog signalling and T cell development. Here we uncover ATM as a novel positive modulator of ITCH E3-ubiquitin ligase activity. A single residue on ITCH protein, S161, which is part of an ATM SQ consensus motif, is required for ATM-dependent activation of ITCH. ATM activity enhances ITCH enzymatic activity, which in turn drives the ubiquitination and degradation of c-FLIP-L and c-Jun, previously identified as ITCH substrates. Importantly, Atm deficient mice show resistance to hepatocyte cell death, similarly to Itch deficient animals, providing in vivo genetic evidence for this circuit. Our data identify ITCH as a novel component of the ATM-dependent signaling pathway and suggest that the impairment of the correct functionality of ITCH caused by Atm deficiency may contribute to the complex clinical features linked to Ataxia Telangiectasia. PMID:23435430

  18. Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N2-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

    PubMed Central

    2012-01-01

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N2-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N2-dGuo (1,N2-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N2-dGuo adduct was incorporated into the 18-mer templates 5′-d(TCATXGAATCCTTCCCCC)-3′ and d(TCACXGAATCCTTCCCCC)-3′, where X = (6S,8R,11S)-HNE-1,N2-dGuo adduct. These differed in the identity of the template 5′-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5′-d(GGGGGAAGGATTC)-3′ or a 14-mer primer 5′-d(GGGGGAAGGATTCC)-3′. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N2-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N2-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N2-dGuo adduct in a sequence-specific manner. If the template 5′-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5′-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5′-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N2-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N2-dGuo adduct, the (6S,8R,11S)-1,N2-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either d

  19. Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA from Streptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA.

    PubMed

    Liu, Guang; Zhang, Zhenyi; Zhao, Gong; Deng, Zixin; Wu, Geng; He, Xinyi

    2015-01-01

    ScoMcrA is a type IV modification-dependent restriction endonuclease found in the model strain Streptomyces coelicolor. Unlike type I, II and III restriction endonucleases, which cleave unmodified DNA, type IV restriction endonucleases cleave modified DNA, including methylated, hydroxymethylated, glucosyl-hydroxymethylated and phosphorothioated DNA. ScoMcrA targets both Dcm-methylated DNA and phosphorothioated DNA, and makes double-strand breaks 16-28 nt away from the modified nucleotides or the phosphorothioate links. However, the mechanism by which ScoMcrA recognizes these two entirely different types of modification remains unclear. In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a=130.19, b=139.36, c=281.01 Å, α=β=γ=90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism.

  20. Systematic approaches to identify E3 ligase substrates

    PubMed Central

    Iconomou, Mary; Saunders, Darren N.

    2016-01-01

    Protein ubiquitylation is a widespread post-translational modification, regulating cellular signalling with many outcomes, such as protein degradation, endocytosis, cell cycle progression, DNA repair and transcription. E3 ligases are a critical component of the ubiquitin proteasome system (UPS), determining the substrate specificity of the cascade by the covalent attachment of ubiquitin to substrate proteins. Currently, there are over 600 putative E3 ligases, but many are poorly characterized, particularly with respect to individual protein substrates. Here, we highlight systematic approaches to identify and validate UPS targets and discuss how they are underpinning rapid advances in our understanding of the biochemistry and biology of the UPS. The integration of novel tools, model systems and methods for target identification is driving significant interest in drug development, targeting various aspects of UPS function and advancing the understanding of a diverse range of disease processes. PMID:27834739

  1. Synthesis, structural characterization, cytotoxicity in vitro, and effect on DNA of sulfate-trans-dichloro-trans-bis(dimethylsulfoxide)-trans-dimethyl-tin(IV)

    NASA Astrophysics Data System (ADS)

    Jiménez-Pérez, Víctor M.; Muñoz-Flores, Blanca M.; Gómez, Alberto; Kharisov, Boris; Santillan, Rosa; Ochoa, María E.; Blanco Jerez, Leonor M.; García, Concepción; Waksman, Noemí; Ramírez, Rosalba

    2014-01-01

    The trans-dichloro-trans-bis(dimethylsulfoxide)-trans-dimethyl-tin(IV) resulted from the reaction of dimethyl-tin(IV) dichloride and sodium sulfate in dimethylsulfoxide (DMSO). The tin compound was characterized by IR, NMR, FE-SEM, EDS, and single crystal X-ray diffraction studies. Suitable DMSO single crystals were obtained after 7 months by slow evaporation at room temperature. The crystal structure has a polymeric arrangement with eight-membered rings as a result of intermolecular coordination SO → Sn. The tin atom has an octahedral distorted geometry where the DMSO molecule is coordinated, and also has an infinite arrangement in 2D. Cytotoxicity against A431 cancer cells and MOLT4 leukemic cells and the DNA interaction of the organotin compound were determined. The organotin sulfate derivative showed low biological activity because of the polymeric structure and the saturated tin coordination capability.

  2. Genetic evidence for an additional function of phage T4 gene 32 protein: interaction with ligase.

    PubMed

    Mosig, G; Breschkin, A M

    1975-04-01

    Gene 32 of bacteriophage T4 is essential for DNA replication, recombination, and repair. In an attempt to clarify the role of the corresponding gene product, we have looked for mutations that specifically inactivate one but not all of its functions and for compensating suppressor mutations in other genes. Here we describe a gene 32 ts mutant that does not produce progeny, but in contrast to an am mutant investigated by others, is capable of some primary and secondary DNA replication and of forming "joint" recombinational intermediates after infection of Escherichia coli B at the restrictive temperature. However, parental and progeny DNA strands are not ligated to covalently linked "recombinant" molecules, and single strands of vegetative DNA do not exceed unit length. Progeny production as well as capacity for covalent linkage in this gene 32 ts mutant are partially restored by additional rII mutations. Suppression by rII depends on functioning host ligase [EC 6.5.1.2; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming, NMN-forming)]. This gene 32 ts mutation (unlike some others) in turn suppresses the characteristic plaque morphology of rII mutants. We conclude that gene 32 protein, in addition to its role in DNA replication and in the formation of "joint" recombinational intermediates, interacts with T4 ligase [EC 6.5.1.1; poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming)] when recombining DNA strands are covalently linked. The protein of the mutant that we describe here is mainly defective in this interaction, thus inactivating T4 ligase in recombination. Suppressing rII mutations facilitate substitution of host ligase. There is suggestive evidence that these interactions occur at the membrane.

  3. Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

    PubMed Central

    Henke, Sarah K.; Cronan, John E.

    2014-01-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

  4. Molecular DNA switches and DNA chips

    NASA Astrophysics Data System (ADS)

    Sabanayagam, Chandran R.; Berkey, Cristin; Lavi, Uri; Cantor, Charles R.; Smith, Cassandra L.

    1999-06-01

    We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 102 - 103 fold discrimination between matched and mismatched sequences.

  5. Twisting Motion Frequency Dependent I-V Characteristics of 102 Base Pairs Poly(dG)-Poly(dC) DNA Molecule

    NASA Astrophysics Data System (ADS)

    Yudiarsah, Efta

    2017-05-01

    The I-V characteristic of 102 base pair Poly(dG)-Poly(dC) DNA molecule have been calculated for several base pairs twisting motion frequencies. The calculation is carried out on doubled-stranded DNA model sandwiched in between two metallic electrodes. The effect is studied by taking into account twisting angle dependent on-site energy and hopping constant in the tight binding Hamiltonian of double-strand DNA model. We use semi-empirical Slater-Koster theory in the twisting angle dependent intra- and inter-strand hopping constant. We consider the temperature dependent sugar-phosphate backbone on-site energy by employing random energy disorder using uniform distribution function. The standard deviation of twisting angle is obtained by assuming that the average kinetic energy of twisting motions is proportional to system temperature. The transfer and scattering matrix methods are used simultaneously in calculating the transmission probability of charge on the molecule. We choose the contacts between molecule and both electrodes such that the main features of transport properties of the molecule do not change much by the presence of metallic electrodes. By assuming the voltage drops symmetrically at the contacts, Landauer-Buttiker Formalism is used in calculating the I-V characteristic of the molecule from transmission probability. The results show that the magnitude of current increases by twisting motion frequency increment. Larger current magnitude increment is observed at higher voltage. The influence of twisting motion frequency on the I-V characteristic is stronger at higher temperature, in the range of considered temperature.

  6. Synthesis, hydrolytic DNA-cleaving activities and cytotoxicities of EDTA analogue-tethered pyrrole-polyamide dimer-based Ce(IV) complexes.

    PubMed

    Yang, Jian-Wei; Lin, Yan-Ling; Dong, Cheng; Zhou, Chun-Qiong; Chen, Jin-Xiang; Wang, Bo; Zhou, Zhong-Zhen; Sun, Bin; Chen, Wen-Hua

    2014-11-24

    Two EDTA analogue-tethered C2-symmetrical dimeric monopyrrole-polyamide 5 and dipyrrole-polyamide 6, and their corresponding Ce(IV) complexes Ce-5 and Ce-6 were synthesized and fully characterized. Agarose gel electrophoresis studies on pBR322 DNA cleavage indicate that complexes Ce-5 and Ce-6 exhibited potent DNA-cleaving activities under physiological conditions. The maximal first-order rate constants (kmax's) were (0.42 ± 0.02) h(-1) for Ce-5 and (0.52 ± 0.02) h(-1) for Ce-6, respectively, suggesting that both complexes catalyzed the cleavage of supercoiled DNA by up to approximately 10(8)-fold. Complex Ce-6 exhibited ca 10-fold higher overall catalytic activity (kmax/KM) than Ce-5, which may be ascribed to its higher DNA-binding affinity. Inhibition experiments and a model study convincingly suggest that both complexes Ce-5 and Ce-6 functioned as hydrolytic DNA-cleavers. In addition, both complexes were found to display moderate inhibitory activity toward A549 and HepG-2 cells. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

    SciTech Connect

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji; An, You Sun; Yi, Jae Youn

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.

  8. Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase

    PubMed Central

    Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart

    2015-01-01

    ATP-dependent RNA ligases are agents of RNA repair that join 3′-OH and 5′-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway—covalent ligase-(lysyl-Nζ)–AMP•Mn2+ intermediate; ligase•ATP•(Mn2+)2 Michaelis complex; and ligase•Mn2+ complex—highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound “catalytic” metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes. PMID:26512110

  9. Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase.

    PubMed

    Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart

    2015-11-10

    ATP-dependent RNA ligases are agents of RNA repair that join 3'-OH and 5'-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl-Nζ)-AMP•Mn(2+) intermediate; ligase•ATP•(Mn(2+))2 Michaelis complex; and ligase•Mn(2+) complex-highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the β- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.

  10. Cullin RING Ligases: Glommed by Glomulin

    PubMed Central

    Hristova, Ventzislava A.; Stringer, Daniel K.; Weissman, Allan M.

    2012-01-01

    Cullin ring ligases (CRLs) constitute the largest group of RING finger ubiquitin ligases. Two recent studies in Molecular Cell describe glomulin as a CRL1 inhibitor that blocks interactions with its ubiquitin-conjugating enzyme (E2) (Duda et al., 2012; Tron et al., 2012). These findings and their significance are discussed. PMID:22883621

  11. Mutational analysis of Trypanosoma brucei RNA editing ligase reveals regions critical for interaction with KREPA2.

    PubMed

    Mehta, Vaibhav; Sen, Rajashree; Moshiri, Houtan; Salavati, Reza

    2015-01-01

    The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1) and the closely related T. brucei RNA Editing Ligase 2 (TbREL2). While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB)-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1) and KREPA1 (for TbREL2). KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441-444) sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.

  12. Synthesis, characterization, in vitro antitumoral investigations and interaction with plasmid pBR322 DNA of R2eddp-platinum(IV) complexes (R = Et, n-Pr).

    PubMed

    Kaluderović, Goran N; Kommera, Harish; Schwieger, Sebastian; Paethanom, Anchan; Kunze, Michael; Schmidt, Harry; Paschke, Reinhard; Steinborn, Dirk

    2009-12-28

    The studies on synthetic, spectroscopic and biological properties of platinum(IV) complexes, [PtCl(4)(R(2)eddp)] (R = Et, 1; n-Pr, 2), containing kappa(2)N,N' bidentate ligands, esters of ethylenediamine-N,N'-di-3-propionic acid (HOOCCH(2)CH(2)NHCH(2)CH(2)NHCH(2)CH(2)COOH, H(2)eddp), are reported. Complexes have been characterized by infrared, (1)H and (13)C NMR spectroscopy and elemental analysis and it was concluded that the coordination of the ligands occurs via nitrogen donor atoms of the ester ligands (R(2)eddp). Cytotoxicity studies were performed for ligand precursors and corresponding platinum(IV) complexes. Although the n-Pr(2)eddp.2HCl itself showed no activity (IC(50) values > 125 microM) in selected cell lines, the activity of complex 2, via apoptotic mode of cell death, has increased significantly for a broad range of cancer cell lines tested in vitro (IC(50) = 8.6-49 microM). As it was found that complexes 1 and 2 are able to interact with pBR322 plasmid DNA, platinum(IV) complexes of this type may act as drugs and pro-drugs.

  13. Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

    PubMed

    Karam, J D; Barker, B

    1971-02-01

    In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is mo