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Catherine T Yan - One of the best experts on this subject based on the ideXlab platform.
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LIG4 is essential for maintaining hsc homeostasis
Blood, 2014Co-Authors: Jihye Park, Robert S Welner, Daniel G Tenen, Catherine T YanAbstract:Blood cells of all lineages are generated from small pools of long-term hematopoietic stem cells (LT-HSCs) that continually replenish throughout life. LT-HSCs regulate the balanced turnover of all mature blood lineages by switching between self-renewal, differentiation and quiescence, thereby maintaining hematopoietic homeostasis in steady state and in response to injury. In any given cell, some DNA damage may remain despite the action of DNA repair processes, including in LT-HSCs. Over time, HSCs lose their long-term capability to self-renew due to misrepair of DNA breaks and increased accumulation of DNA damage, resulting in loss of regenerative plasticity and immune fitness. The accrual of DNA damage is the principal factor that contributes to functional decline in HSC renewal and in the immune system during ageing. In recent studies, the capacity of aging HSCs to self-renew is shown to be dependent on DNA repair pathways, with non-homologous end-joining (NHEJ) as the principle pathway implicated in DNA repair in quiescent HSCs from ex-vivo cell-based assays. Although NHEJ in particular has been implicated in this process in LT-HSCs, there has so far been very little evidence of this activity in vivo. DNA Ligase IV (LIG4), which catalyzes the end-ligation of broken DNA ends mediated by NHEJ, has no known functions outside of NHEJ. Because a deficiency in LIG4 in mice is embryonic lethal, here we assessed the role of LIG4 in HSC homeostasis by assaying HSC functions in a knockin mouse model of a hypomorphic homozygous R278H mutation in LIG4 that had been identified in the first DNA LIG4 Syndrome patient. The R278H mutation significantly impairs the end-ligation function of the LIG4 protein, and mice homozygous for the R278H mutation (LIG4R/R) showed diminished DSB repair capacity and age-dependent lymphopenia that implicated potential HSC defects. Consistent with a defect in NHEJ, we show the LIG4 R278H mutation severely limited HSC self-renewal. LIG4R/R HSC reconstitutions were skewed towards the myeloid lineage and resulted in severely reduced chimerism, confirming the capacity of HSCs to self-renew requires functional DNA repair. Next, we examined if there is increased DNA damage with/without ionizing irradiation (IR). LIG4R/R LT-HSCs showed an increase in reactive oxygen species (ROS), abnormal cycling and increased apoptosis from accumulated DNA damage in steady state and slow DNA double strand breaks (DSBs) repair kinetics in response to low dose IR because of improper LIG4 function. This led us to check the LT-HSC pool more carefully. It has been shown that the HSC pool is intact and phenotypically increased with age. Strikingly, we found that the HSCs in both young and old LIG4R/R mice are markedly reduced to 20% of wild-type levels. The severe LT-HSC reduction and lethality of disease in LIG4R/Rmice was completely rescued by transplantation with wild type bone marrow. These evidences support the notion of a critical role for LIG4 in maintenance of the LT-HSC pool. In a recent study, it was reported that the steady state pool of murine adult LT-HSCs can be further distinguished into quiescent (~20%) and variably cycling (~80%) populations. Since LT-HSCs in young LIG4R/R mice are maintained in steady state at 20% of WT HSCs, we hypothesized that the reduced pool of LT-HSCs in the LIG4R/R mice is caused by the loss of cycling LT-HSCs that continually replenish blood lineages during aging. To compare these populations, microarray analysis was done on the pool of WT and LIG4R/R LT-HSCs, versus sorted populations of quiescent and variably cycling LT-HSCs. Microarray analysis clearly showed that the LIG4R/RLT-HSCs correlated with the quiescent LT-HSCs, indicating NHEJ regulates the homeostasis of the faster cycling LT-HSC pool. Our study suggest that the slowest cycling LT-HSCs serve to replenish the overall LT-HSC pool and HSC homeostasis is maintained by capacity of faster cycling LT-HSC pool to revert to quiescence in response to stress/injury. Additionally, defective NHEJ depletes the faster cycling LT-HSC pool and underlies early HSC exhaustion in LIG4R/R mice. Our findings demonstrate for the first time a physiological role for LIG4 in the maintenance of HSC homeostasis. Disclosures No relevant conflicts of interest to declare.
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LIG4 modulates p53 p21 checkpoints and inhibits a ej during end joining repair phase of igh class switching lym6p 770
Journal of Immunology, 2014Co-Authors: Jihye Park, Robert S Welner, Daniel G Tenen, Catherine T YanAbstract:Mutations in the non-homologous end-joining DNA repair protein DNA Ligase IV (LIG4) causes a human syndrome of immunodeficiency and radiosensitivity. We previously described knock-in mice with a homozygous R278H mutation in LIG4 (LIG4R/R) that displayed severely leaky B lymphocyte development, resulting in near lack of mature B cells yet only a partial block in IgH class switch recombination (CSR). Here we show that diminished DNA ligase 4 activity, resulting in slower kinetics of DNA double strand break (DSB) repair and attenuation of p53-dependent DNA double strand break (DSB) response, to underlie the partial CSR block in the LIG4R/R activated B cells. Analysis of p53 levels in activated LIG4R/R B cells revealed increased proteosome mediated p53 decay and p21 activation, indicative of slower repair kinetics, effects mimicked in the human LIG4 R278H 180BR cell line. Concurrent p53 inactivation had little effect on the frequency of IgH breaks; but substantially slowed the cell cycle progression of activated LIG4R/Rp53-/- B cells, and failed to promote the development of B-lymphomas that recurrently developed in murine LIG4-/-p53-/- settings. Analysis LIG4R/R CSR junctions also revealed the predominance of NHEJ instead of NHEJ-independent A-EJ in their generation. Hence NHEJ maintains genomic stability by modulating the p53 DNA damage response and inhibiting A-EJ. These findings provide an explanation for the complex B-lymphoid phenotypes of the human LIG4 Syndrome.
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hypomorphic LIG4 impacts on igh class switching and translocations
Blood, 2010Co-Authors: Logan Troppito, Parames Muniandy, Sabrine Hedouin, Joshua Silva, Lauren Feldman, Catherine T YanAbstract:Abstract Abstract 3373 The DNA Ligase IV (LIG4) protein is an essential component of the non-homologous end-joining (NHEJ) repair pathway that plays a key role in the repair of general DNA double strand breaks (DSBs) and those generated during V(D)J recombination and IgH Class Switch Recombination (CSR) in B lymphocytes. Hypomorphic mutations in the LIG4 gene in humans underlies the DNA LIG4 Syndrome, which is associated with radiosensitivity, developmental delay, growth defects, facial dysmorphism and variable degrees of immunodeficiencies. We recently described a mouse model carrying a specific LIG4 hypomorphic mutation in both copies of the LIG4 coding exon that generates an arginine to histidine amino acid #278 (R278H) replacement that corresponds to the first LIG4 mutation reported in humans. Our studies revealed that similar to human patients, the phenotype of homozygous mutant mice LIG4R278H/R278H (LIG4R/R) includes growth retardation, significant decreased life span, severe cellular sensitivity to ionizing radiation, and a severe but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID, and high levels of genomic instability associated with increased incidence of thymic and peripheral T lymphomas. Severe defects in B lymphocyte developmental functions include repertoire restriction during aging, spontaneous low affinity antibody production and inability mount high affinity antibody responses. Our studies now reveal that introduction of IgH and IgL knock-in (HL) variable region exon into the R/R mice leads to significant increase in mature B lymphocytes in peripheral lymphoid organs, directly demonstrating that the overall severe reduction in B lymphocytes in the R/R mice is in part due to a block in their developmental progression. We further observe a mild reduction in IgH class switching in the in vitro CSR activated R/RHL B lymphocytes, accompanied by an unusual increase in IgH specific DSBs and translocations, at levels significantly higher than what we previously observed in LIG4 or XRCC4 deficient peripheral B cells, and lower than expected general DSBs. The implications of our findings in the context of B lymphocyte development and CSR, potential altered utilization of alternative end-joining DSB repair mechanisms and on B lymphomagenesis will be discussed. Disclosures: No relevant conflicts of interest to declare.
Judy L Bolton - One of the best experts on this subject based on the ideXlab platform.
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induction of nad p h quinone oxidoreductase 1 nqo1 by glycyrrhiza species used for women s health differential effects of the michael acceptors isoliquiritigenin and licochalcone a
Chemical Research in Toxicology, 2015Co-Authors: Atieh Hajirahimkhan, Charlotte Simmler, Shaonong Chen, Guido F Pauli, Birgit M Dietz, Huali Dong, Daniel D Lantvit, Dejan Nikolic, Richard B Van Breemen, Judy L BoltonAbstract:For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human he...
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differential effects of glycyrrhiza species on genotoxic estrogen metabolism licochalcone a downregulates p450 1b1 whereas isoliquiritigenin stimulates it
Chemical Research in Toxicology, 2015Co-Authors: Tareisha L Dunlap, Shuai Wang, Charlotte Simmler, Shaonong Chen, Guido F Pauli, Birgit M Dietz, Judy L BoltonAbstract:Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to...
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differential effects of glycyrrhiza species on genotoxic estrogen metabolism licochalcone a downregulates p450 1b1 whereas isoliquiritigenin stimulates it
Chemical Research in Toxicology, 2015Co-Authors: Tareisha L Dunlap, Shuai Wang, Charlotte Simmler, Shaonong Chen, Guido F Pauli, Birgit M Dietz, Judy L BoltonAbstract:Estrogen chemical carcinogenesis involves 4-hydroxylation of estrone/estradiol (E1/E2) by P450 1B1, generating catechol and quinone genotoxic metabolites that cause DNA mutations and initiate/promote breast cancer. Inflammation enhances this effect by upregulating P450 1B1. The present study tested the three authenticated medicinal species of licorice [Glycyrrhiza glabra (GG), G. uralensis (GU), and G. inflata (GI)] used by women as dietary supplements for their anti-inflammatory activities and their ability to modulate estrogen metabolism. The pure compounds, liquiritigenin (LigF), its chalcone isomer isoliquiritigenin (LigC), and the GI-specific licochalcone A (LicA) were also tested. The licorice extracts and compounds were evaluated for anti-inflammatory activity by measuring inhibition of iNOS activity in macrophage cells: GI ≫ GG > GU and LigC ≅ LicA ≫ LigF. The Michael acceptor chalcone, LicA, is likely responsible for the anti-inflammatory activity of GI. A sensitive LC-MS/MS assay was employed to quantify estrogen metabolism by measuring 2-MeOE1 as nontoxic and 4-MeOE1 as genotoxic biomarkers in the nontumorigenic human mammary epithelial cell line, MCF-10A. GG, GU, and LigC increased 4-MeOE1, whereas GI and LicA inhibited 2- and 4-MeOE1 levels. GG, GU (5 μg/mL), and LigC (1 μM) also enhanced P450 1B1 expression and activities, which was further increased by inflammatory cytokines (TNF-α and IFN-γ). LicA (1, 10 μM) decreased cytokine- and TCDD-induced P450 1B1 gene expression and TCDD-induced xenobiotic response element luciferase reporter (IC50 = 12.3 μM), suggesting an antagonistic effect on the aryl hydrocarbon receptor, which regulates P450 1B1. Similarly, GI (5 μg/mL) reduced cytokine- and TCDD-induced P450 1B1 gene expression. Collectively, these data suggest that, of the three licorice species that are used in botanical supplements, GI represents the most promising chemopreventive licorice extract for women's health. Additionally, the differential effects of the Glycyrrhiza species on estrogen metabolism emphasize the importance of standardization of botanical supplements to species-specific bioactive compounds.
Jose Henrique Pereira - One of the best experts on this subject based on the ideXlab platform.
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structural and biochemical characterization of the early and late enzymes in the lignin β aryl ether cleavage pathway from sphingobium sp syk 6
Journal of Biological Chemistry, 2016Co-Authors: Jose Henrique Pereira, Richard A Heins, Daniel L Gall, Ryan P Mcandrew, Kai DengAbstract:There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.
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structural basis of stereospecificity in the bacterial enzymatic cleavage of β aryl ether bonds in lignin
Journal of Biological Chemistry, 2016Co-Authors: Jose Henrique Pereira, Richard A Heins, Daniel L Gall, Ryan P Mcandrew, Kate E Helmich, Craig A BingmanAbstract:Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
Jihye Park - One of the best experts on this subject based on the ideXlab platform.
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the dna ligase iv syndrome r278h mutation impairs b lymphopoiesis via error prone nonhomologous end joining
Journal of Immunology, 2016Co-Authors: Jihye Park, Robert S Welner, Logan Troppito, Meiyee Chan, Philipp B Staber, Daniel G TenenAbstract: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.
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LIG4 is essential for maintaining hsc homeostasis
Blood, 2014Co-Authors: Jihye Park, Robert S Welner, Daniel G Tenen, Catherine T YanAbstract:Blood cells of all lineages are generated from small pools of long-term hematopoietic stem cells (LT-HSCs) that continually replenish throughout life. LT-HSCs regulate the balanced turnover of all mature blood lineages by switching between self-renewal, differentiation and quiescence, thereby maintaining hematopoietic homeostasis in steady state and in response to injury. In any given cell, some DNA damage may remain despite the action of DNA repair processes, including in LT-HSCs. Over time, HSCs lose their long-term capability to self-renew due to misrepair of DNA breaks and increased accumulation of DNA damage, resulting in loss of regenerative plasticity and immune fitness. The accrual of DNA damage is the principal factor that contributes to functional decline in HSC renewal and in the immune system during ageing. In recent studies, the capacity of aging HSCs to self-renew is shown to be dependent on DNA repair pathways, with non-homologous end-joining (NHEJ) as the principle pathway implicated in DNA repair in quiescent HSCs from ex-vivo cell-based assays. Although NHEJ in particular has been implicated in this process in LT-HSCs, there has so far been very little evidence of this activity in vivo. DNA Ligase IV (LIG4), which catalyzes the end-ligation of broken DNA ends mediated by NHEJ, has no known functions outside of NHEJ. Because a deficiency in LIG4 in mice is embryonic lethal, here we assessed the role of LIG4 in HSC homeostasis by assaying HSC functions in a knockin mouse model of a hypomorphic homozygous R278H mutation in LIG4 that had been identified in the first DNA LIG4 Syndrome patient. The R278H mutation significantly impairs the end-ligation function of the LIG4 protein, and mice homozygous for the R278H mutation (LIG4R/R) showed diminished DSB repair capacity and age-dependent lymphopenia that implicated potential HSC defects. Consistent with a defect in NHEJ, we show the LIG4 R278H mutation severely limited HSC self-renewal. LIG4R/R HSC reconstitutions were skewed towards the myeloid lineage and resulted in severely reduced chimerism, confirming the capacity of HSCs to self-renew requires functional DNA repair. Next, we examined if there is increased DNA damage with/without ionizing irradiation (IR). LIG4R/R LT-HSCs showed an increase in reactive oxygen species (ROS), abnormal cycling and increased apoptosis from accumulated DNA damage in steady state and slow DNA double strand breaks (DSBs) repair kinetics in response to low dose IR because of improper LIG4 function. This led us to check the LT-HSC pool more carefully. It has been shown that the HSC pool is intact and phenotypically increased with age. Strikingly, we found that the HSCs in both young and old LIG4R/R mice are markedly reduced to 20% of wild-type levels. The severe LT-HSC reduction and lethality of disease in LIG4R/Rmice was completely rescued by transplantation with wild type bone marrow. These evidences support the notion of a critical role for LIG4 in maintenance of the LT-HSC pool. In a recent study, it was reported that the steady state pool of murine adult LT-HSCs can be further distinguished into quiescent (~20%) and variably cycling (~80%) populations. Since LT-HSCs in young LIG4R/R mice are maintained in steady state at 20% of WT HSCs, we hypothesized that the reduced pool of LT-HSCs in the LIG4R/R mice is caused by the loss of cycling LT-HSCs that continually replenish blood lineages during aging. To compare these populations, microarray analysis was done on the pool of WT and LIG4R/R LT-HSCs, versus sorted populations of quiescent and variably cycling LT-HSCs. Microarray analysis clearly showed that the LIG4R/RLT-HSCs correlated with the quiescent LT-HSCs, indicating NHEJ regulates the homeostasis of the faster cycling LT-HSC pool. Our study suggest that the slowest cycling LT-HSCs serve to replenish the overall LT-HSC pool and HSC homeostasis is maintained by capacity of faster cycling LT-HSC pool to revert to quiescence in response to stress/injury. Additionally, defective NHEJ depletes the faster cycling LT-HSC pool and underlies early HSC exhaustion in LIG4R/R mice. Our findings demonstrate for the first time a physiological role for LIG4 in the maintenance of HSC homeostasis. Disclosures No relevant conflicts of interest to declare.
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LIG4 modulates p53 p21 checkpoints and inhibits a ej during end joining repair phase of igh class switching lym6p 770
Journal of Immunology, 2014Co-Authors: Jihye Park, Robert S Welner, Daniel G Tenen, Catherine T YanAbstract:Mutations in the non-homologous end-joining DNA repair protein DNA Ligase IV (LIG4) causes a human syndrome of immunodeficiency and radiosensitivity. We previously described knock-in mice with a homozygous R278H mutation in LIG4 (LIG4R/R) that displayed severely leaky B lymphocyte development, resulting in near lack of mature B cells yet only a partial block in IgH class switch recombination (CSR). Here we show that diminished DNA ligase 4 activity, resulting in slower kinetics of DNA double strand break (DSB) repair and attenuation of p53-dependent DNA double strand break (DSB) response, to underlie the partial CSR block in the LIG4R/R activated B cells. Analysis of p53 levels in activated LIG4R/R B cells revealed increased proteosome mediated p53 decay and p21 activation, indicative of slower repair kinetics, effects mimicked in the human LIG4 R278H 180BR cell line. Concurrent p53 inactivation had little effect on the frequency of IgH breaks; but substantially slowed the cell cycle progression of activated LIG4R/Rp53-/- B cells, and failed to promote the development of B-lymphomas that recurrently developed in murine LIG4-/-p53-/- settings. Analysis LIG4R/R CSR junctions also revealed the predominance of NHEJ instead of NHEJ-independent A-EJ in their generation. Hence NHEJ maintains genomic stability by modulating the p53 DNA damage response and inhibiting A-EJ. These findings provide an explanation for the complex B-lymphoid phenotypes of the human LIG4 Syndrome.
Stuart A Macneill - One of the best experts on this subject based on the ideXlab platform.
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atp and nad dependent dna ligases share an essential function in the halophilic archaeon haloferax volcanii
Molecular Microbiology, 2006Co-Authors: An Zhao, Fiona C Gray, Stuart A MacneillAbstract:Summary DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.