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Robert J. G. Haché - One of the best experts on this subject based on the ideXlab platform.
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activation of parp 1 in response to bleomycin depends on the Ku Antigen and protein phosphatase 5
Oncogene, 2010Co-Authors: F Dong, S Soubeyrand, Robert J. G. HachéAbstract:Poly (ADP-ribose) polymerase-1 (PARP-1) has an important role in the cellular response to a broad spectrum of DNA lesions. PARP-1 is strongly activated in response to double-stranded DNA breaks (DSBs), yet its contribution to the DSB response is poorly understood. Here we used bleomycin, a radiomimetic that generates DSBs with high specificity to focus on the response of PARP-1 to DSBs. We report that the induction of PARP-1 activity by bleomycin depends on the Ku Antigen, a nonhomologous-DNA-End-Joining factor and protein phosphatase 5 (PP5). PARP-1 activation in response to bleomycin was reduced over 10-fold in Ku-deficient cells, whereas its activation in response to U.V. was unaffected. PARP-1 activation was rescued by reexpression of Ku, but was refractory to manipulation of DNA-dependent protein kinase or ATM. Similarly, PARP-1 activation subsequent to bleomycin was reduced 2-fold on ablation of PP5 and was increased 5-fold when PP5 was overexpressed. PP5 seemed to act directly on PARP-1, as its basal phosphorylation was reduced on overexpression of PP5, and PP5 dephosphorylated PARP-1 in vitro. These results highlight the functional importance of Ku Antigen and PP5 for PARP-1 activity subsequent to DSBs.
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down regulation of histone h2b by dna dependent protein kinase in response to dna damage through modulation of octamer transcription factor 1
Cancer Research, 2003Co-Authors: Caroline Schildpoulter, Amy Shih, Nicholas C Yarymowich, Robert J. G. HachéAbstract:Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku Antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.
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differential dna binding of Ku Antigen determines its involvement in dna replication
DNA and Cell Biology, 2003Co-Authors: Caroline Schildpoulter, Ward Giffin, Diamanto Matheos, Olivia Novac, Bo Cui, Marcia T Ruiz, Gerald B Price, Maria Zannishadjopoulos, Robert J. G. HachéAbstract:Ku Antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku Antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.
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evidence implicating Ku Antigen as a structural factor in rna polymerase ii mediated transcription
Gene, 2003Co-Authors: Jesse Bertinato, Caroline Schildpoulter, Julianna J Tomlinson, Robert J. G. HachéAbstract:Ku Antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA. We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku. In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments. Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location. Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70. The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography. These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme.
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the binding of Ku Antigen to homeodomain proteins promotes their phosphorylation by dna dependent protein kinase
Journal of Biological Chemistry, 2001Co-Authors: Caroline Schildpoulter, Louise Pope, Ward Giffin, Jeff C. Kochan, Johnny K. Ngsee, Maya Traykovaandonova, Robert J. G. HachéAbstract:The Ku Antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.
Jacques Piette - One of the best experts on this subject based on the ideXlab platform.
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Ku Antigen is required to relieve g2 arrest caused by inhibition of dna topoisomerase ii activity by the bisdioxopiperazine icrf 193
Oncogene, 2001Co-Authors: Purificacion Munoz, Fabienne Baus, Jacques PietteAbstract:Ku Antigen is necessary for DNA double-strand break (DSB) repair through its ability to bind DNA ends with high affinity and to recruit the catalytic subunit of DNA-PK to the DSBs. Ku-deficient cells are hypersensitive to agents causing DSBs in DNA but also to the DNA topoisomerase II (topo II) inhibitor ICRF-193, which does not induce DSBs. This suggests a new role of Ku Antigen, that is independent of DSB repair by DNA-PK. Here we characterize the basis for the hypersensitivity of Ku-deficient cells to ICRF-193. Chromosome condensation and segregation, which are dependent on topo II, but also the catalytic activity of topo II in late S-G2 were inhibited to a comparable extent when ICRF-193 was applied to Ku-deficient cells or wild-type cells. However, mutant cells arrested in G2 by ICRF-193 treatment were unable to progress into M phase upon drug removal, although drug-trapped topo II complexes were removed from DNA and the two isoforms of topo II recovered their catalytic activity as in wild-type cells. The reversibility of G2 arrest was recovered by complementation of mutant cells with a human Ku86 cDNA. Notably, chromosome condensation was abnormal in Ku-deficient cells after suppression of the G2 arrest by caffeine, even in the absence of ICRF-193. These results reflect the involvement of Ku-Antigen in the cellular response to topo II inhibition, more particularly in relieving G2 arrest caused by topo II inhibition in late S/G2 and the subsequent recovery of chromosome condensation.
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hypersensitivity of Ku deficient cells toward the dna topoisomerase ii inhibitor icrf 193 suggests a novel role for Ku Antigen during the g2 and m phases of the cell cycle
Molecular and Cellular Biology, 1998Co-Authors: Purificacion Munoz, Jeanmarie Blanchard, Massgorzata Z Zdzienicka, Jacques PietteAbstract:Ku is an abundant nuclear protein originally identified as an autoAntigen recognized by sera from patients with autoimmune diseases (47). The Ku Antigen is a heterodimer comprised of 86- and 70-kDa subunits (Ku86 and Ku70) which interacts preferentially with free DNA ends or particular DNA structures such as hairpins or gaps (46–48). Ku is associated with a catalytic subunit of 450 kDa (DNA-PKCS), forming the DNA-dependent protein kinase (DNA-PK). It is presumed that the Ku Antigen tethers the serine-threonine kinase activity of this enzyme to DNA ends, allowing phosphorylation of nearby substrates (22, 28). Accordingly, in vitro phosphorylation of a large number of proteins by DNA-PK is stimulated in the presence of double-strand breaks (DSB) (12, 43; reviewed in reference 3). A series of genetic and biochemical data have converged to establish the crucial role of DNA-PK in DSB repair as well as in V(D)J recombination of immunoglobulin and T-cell receptor genes (reviewed in references 35, 69, and 75). Defective DSB repair and V(D)J recombination in X-ray-sensitive rodent cell lines of the complementation group for XRCC5 were rescued by transfection of the human Ku86 cDNA (9, 57, 59), and mutations that are responsible for the X-ray sensitivity of these cells were mapped to the Ku86 gene, demonstrating unambiguously that Ku86 is the product of XRCC5 (25). An additional role for DNA-PK in DNA replication has been suggested by its ability to phosphorylate replication protein A (10, 52). Furthermore, a role in transcription is indicated by the ability of DNA-PK to inhibit RNA polymerase I activity (39, 41), as well as to modulate the activity of transcription factors through phosphorylation (27). Recent data provide evidence that the Ku Antigen possesses additional functions that are not directly related to its role as part of the DNA-PK enzyme. Indeed, Ku86-null mice display considerable growth defects in addition to an absence of T- and B-lymphocyte maturation (51, 77). Such growth retardation is not observed in SCID mice or foals, which are defective in DNA-PKCS (8, 51, 70). Interestingly, Ku Antigen has been shown to possess in vitro intrinsic DNA-dependent ATPase and helicase activity (11, 62). DNA topoisomerase II catalyzes topological changes in DNA that are essential for normal cell cycle progression. The enzyme can relax supercoiled DNA and can also knot or unknot DNA as well as catenate and decatenate closed circular DNA by passing duplex DNA through an enzyme-mediated DNA gate in an ATP-dependent manner (reviewed in reference 65). Budding yeast topoisomerase II is an essential enzyme for disentangling sister chromatids after DNA replication (31), while the fission yeast enzyme is also involved in chromosome condensation during metaphase (63). Additional evidence for a role of DNA topoisomerase II in chromosome condensation has been provided by in vitro experiments using extracts from Xenopus eggs (2, 30, 50). Although definitive proof that DNA topoisomerase II is required for disentanglement and condensation of sister chromatids in mammalian cells has not been obtained, due to the absence of topoisomerase II mutants, mammalian cells treated with ICRF-193, a novel noncleavable complex-stabilizing type topoisomerase II inhibitor, do not undergo normal chromosome condensation (5, 16, 21, 34). Two topoisomerase II isoforms, designated topoisomerase IIα and topoisomerase IIβ, exist in mammalian cells. DNA topoisomerase IIα expression is cell cycle dependent and peaks during the G2 and M phases (38, 45, 71). The expression pattern of the β isoform was variably reported to remain constant throughout the cell cycle (38, 71) or to increase during the S, G2, and M phases (45). DNA topoisomerase IIα is a major constituent of the nuclear scaffold and has been implicated directly in the organization of metaphase chromosomes (13, 23, 26, 45). Less is known about the role of the β isoform, which is nucleoplasmic during interphase and diffuses into the cytosol during mitosis (45). Although DNA topoisomerase IIα clearly plays a catalytic role in decatenation of linked chromatids, its function in chromosome condensation may be stoichiometric (1, 6, 45). DNA topoisomerase II preferentially associates with AT-rich sequences present in the scaffold-associated regions, which are candidate DNA elements for defining the bases of chromatin loops and serving as cis elements of chromosome dynamics (1, 18, 58). Moreover, purified enzyme has been shown to multimerize in vitro, and this aggregation is stimulated by phosphorylation of its C terminus (64). It is known that X-ray-sensitive cell lines deficient in subunits of the DNA-PK enzyme are hypersensitive to DNA topoisomerase II inhibitors such as etoposide, which stabilize topoisomerase II-DNA cleavage complexes and thus concomitantly induce DSB (44). This hypersensitivity has generally been ascribed to deficiency of these mutant cell lines in DSB repair (29, 36, 66). In contrast to etoposide, ICRF-193 inhibits DNA topoisomerase II activity without inducing any DSB (21, 61). Thus, analysis of cells treated with ICRF-193 allows the effects of DNA topoisomerase II inhibition to be separated from those due to the introduction of DSB. We now demonstrate that topoisomerase II-mediated functions are inhibited at significantly lower doses of ICRF-193 in Ku86-deficient cell lines than in wild-type cells. This difference was not observed when DNA-PKCS-deficient cells were analyzed. Our data indicate a novel role for Ku Antigen in the G2 and M phases of the cell cycle, one that appears independent of its participation in DSB repair by DNA-PK.
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Hypersensitivity of Ku-deficient cells toward the DNA topoisomerase II inhibitor ICRF-193 suggests a novel role for Ku Antigen during the G2 and M phases of the cell cycle
Molecular and Cellular Biology, 1998Co-Authors: Purificacion Munoz, Massgorzata Z Zdzienicka, J. M. Blanchard, Jacques PietteAbstract:Ku Antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIalpha or -beta. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G2/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku Antigen in the G2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.
Purificacion Munoz - One of the best experts on this subject based on the ideXlab platform.
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Ku Antigen is required to relieve g2 arrest caused by inhibition of dna topoisomerase ii activity by the bisdioxopiperazine icrf 193
Oncogene, 2001Co-Authors: Purificacion Munoz, Fabienne Baus, Jacques PietteAbstract:Ku Antigen is necessary for DNA double-strand break (DSB) repair through its ability to bind DNA ends with high affinity and to recruit the catalytic subunit of DNA-PK to the DSBs. Ku-deficient cells are hypersensitive to agents causing DSBs in DNA but also to the DNA topoisomerase II (topo II) inhibitor ICRF-193, which does not induce DSBs. This suggests a new role of Ku Antigen, that is independent of DSB repair by DNA-PK. Here we characterize the basis for the hypersensitivity of Ku-deficient cells to ICRF-193. Chromosome condensation and segregation, which are dependent on topo II, but also the catalytic activity of topo II in late S-G2 were inhibited to a comparable extent when ICRF-193 was applied to Ku-deficient cells or wild-type cells. However, mutant cells arrested in G2 by ICRF-193 treatment were unable to progress into M phase upon drug removal, although drug-trapped topo II complexes were removed from DNA and the two isoforms of topo II recovered their catalytic activity as in wild-type cells. The reversibility of G2 arrest was recovered by complementation of mutant cells with a human Ku86 cDNA. Notably, chromosome condensation was abnormal in Ku-deficient cells after suppression of the G2 arrest by caffeine, even in the absence of ICRF-193. These results reflect the involvement of Ku-Antigen in the cellular response to topo II inhibition, more particularly in relieving G2 arrest caused by topo II inhibition in late S/G2 and the subsequent recovery of chromosome condensation.
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hypersensitivity of Ku deficient cells toward the dna topoisomerase ii inhibitor icrf 193 suggests a novel role for Ku Antigen during the g2 and m phases of the cell cycle
Molecular and Cellular Biology, 1998Co-Authors: Purificacion Munoz, Jeanmarie Blanchard, Massgorzata Z Zdzienicka, Jacques PietteAbstract:Ku is an abundant nuclear protein originally identified as an autoAntigen recognized by sera from patients with autoimmune diseases (47). The Ku Antigen is a heterodimer comprised of 86- and 70-kDa subunits (Ku86 and Ku70) which interacts preferentially with free DNA ends or particular DNA structures such as hairpins or gaps (46–48). Ku is associated with a catalytic subunit of 450 kDa (DNA-PKCS), forming the DNA-dependent protein kinase (DNA-PK). It is presumed that the Ku Antigen tethers the serine-threonine kinase activity of this enzyme to DNA ends, allowing phosphorylation of nearby substrates (22, 28). Accordingly, in vitro phosphorylation of a large number of proteins by DNA-PK is stimulated in the presence of double-strand breaks (DSB) (12, 43; reviewed in reference 3). A series of genetic and biochemical data have converged to establish the crucial role of DNA-PK in DSB repair as well as in V(D)J recombination of immunoglobulin and T-cell receptor genes (reviewed in references 35, 69, and 75). Defective DSB repair and V(D)J recombination in X-ray-sensitive rodent cell lines of the complementation group for XRCC5 were rescued by transfection of the human Ku86 cDNA (9, 57, 59), and mutations that are responsible for the X-ray sensitivity of these cells were mapped to the Ku86 gene, demonstrating unambiguously that Ku86 is the product of XRCC5 (25). An additional role for DNA-PK in DNA replication has been suggested by its ability to phosphorylate replication protein A (10, 52). Furthermore, a role in transcription is indicated by the ability of DNA-PK to inhibit RNA polymerase I activity (39, 41), as well as to modulate the activity of transcription factors through phosphorylation (27). Recent data provide evidence that the Ku Antigen possesses additional functions that are not directly related to its role as part of the DNA-PK enzyme. Indeed, Ku86-null mice display considerable growth defects in addition to an absence of T- and B-lymphocyte maturation (51, 77). Such growth retardation is not observed in SCID mice or foals, which are defective in DNA-PKCS (8, 51, 70). Interestingly, Ku Antigen has been shown to possess in vitro intrinsic DNA-dependent ATPase and helicase activity (11, 62). DNA topoisomerase II catalyzes topological changes in DNA that are essential for normal cell cycle progression. The enzyme can relax supercoiled DNA and can also knot or unknot DNA as well as catenate and decatenate closed circular DNA by passing duplex DNA through an enzyme-mediated DNA gate in an ATP-dependent manner (reviewed in reference 65). Budding yeast topoisomerase II is an essential enzyme for disentangling sister chromatids after DNA replication (31), while the fission yeast enzyme is also involved in chromosome condensation during metaphase (63). Additional evidence for a role of DNA topoisomerase II in chromosome condensation has been provided by in vitro experiments using extracts from Xenopus eggs (2, 30, 50). Although definitive proof that DNA topoisomerase II is required for disentanglement and condensation of sister chromatids in mammalian cells has not been obtained, due to the absence of topoisomerase II mutants, mammalian cells treated with ICRF-193, a novel noncleavable complex-stabilizing type topoisomerase II inhibitor, do not undergo normal chromosome condensation (5, 16, 21, 34). Two topoisomerase II isoforms, designated topoisomerase IIα and topoisomerase IIβ, exist in mammalian cells. DNA topoisomerase IIα expression is cell cycle dependent and peaks during the G2 and M phases (38, 45, 71). The expression pattern of the β isoform was variably reported to remain constant throughout the cell cycle (38, 71) or to increase during the S, G2, and M phases (45). DNA topoisomerase IIα is a major constituent of the nuclear scaffold and has been implicated directly in the organization of metaphase chromosomes (13, 23, 26, 45). Less is known about the role of the β isoform, which is nucleoplasmic during interphase and diffuses into the cytosol during mitosis (45). Although DNA topoisomerase IIα clearly plays a catalytic role in decatenation of linked chromatids, its function in chromosome condensation may be stoichiometric (1, 6, 45). DNA topoisomerase II preferentially associates with AT-rich sequences present in the scaffold-associated regions, which are candidate DNA elements for defining the bases of chromatin loops and serving as cis elements of chromosome dynamics (1, 18, 58). Moreover, purified enzyme has been shown to multimerize in vitro, and this aggregation is stimulated by phosphorylation of its C terminus (64). It is known that X-ray-sensitive cell lines deficient in subunits of the DNA-PK enzyme are hypersensitive to DNA topoisomerase II inhibitors such as etoposide, which stabilize topoisomerase II-DNA cleavage complexes and thus concomitantly induce DSB (44). This hypersensitivity has generally been ascribed to deficiency of these mutant cell lines in DSB repair (29, 36, 66). In contrast to etoposide, ICRF-193 inhibits DNA topoisomerase II activity without inducing any DSB (21, 61). Thus, analysis of cells treated with ICRF-193 allows the effects of DNA topoisomerase II inhibition to be separated from those due to the introduction of DSB. We now demonstrate that topoisomerase II-mediated functions are inhibited at significantly lower doses of ICRF-193 in Ku86-deficient cell lines than in wild-type cells. This difference was not observed when DNA-PKCS-deficient cells were analyzed. Our data indicate a novel role for Ku Antigen in the G2 and M phases of the cell cycle, one that appears independent of its participation in DSB repair by DNA-PK.
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Hypersensitivity of Ku-deficient cells toward the DNA topoisomerase II inhibitor ICRF-193 suggests a novel role for Ku Antigen during the G2 and M phases of the cell cycle
Molecular and Cellular Biology, 1998Co-Authors: Purificacion Munoz, Massgorzata Z Zdzienicka, J. M. Blanchard, Jacques PietteAbstract:Ku Antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIalpha or -beta. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G2/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku Antigen in the G2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.
Caroline Schildpoulter - One of the best experts on this subject based on the ideXlab platform.
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down regulation of histone h2b by dna dependent protein kinase in response to dna damage through modulation of octamer transcription factor 1
Cancer Research, 2003Co-Authors: Caroline Schildpoulter, Amy Shih, Nicholas C Yarymowich, Robert J. G. HachéAbstract:Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku Antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.
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differential dna binding of Ku Antigen determines its involvement in dna replication
DNA and Cell Biology, 2003Co-Authors: Caroline Schildpoulter, Ward Giffin, Diamanto Matheos, Olivia Novac, Bo Cui, Marcia T Ruiz, Gerald B Price, Maria Zannishadjopoulos, Robert J. G. HachéAbstract:Ku Antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku Antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.
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evidence implicating Ku Antigen as a structural factor in rna polymerase ii mediated transcription
Gene, 2003Co-Authors: Jesse Bertinato, Caroline Schildpoulter, Julianna J Tomlinson, Robert J. G. HachéAbstract:Ku Antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA. We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku. In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments. Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location. Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70. The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography. These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme.
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the binding of Ku Antigen to homeodomain proteins promotes their phosphorylation by dna dependent protein kinase
Journal of Biological Chemistry, 2001Co-Authors: Caroline Schildpoulter, Louise Pope, Ward Giffin, Jeff C. Kochan, Johnny K. Ngsee, Maya Traykovaandonova, Robert J. G. HachéAbstract:The Ku Antigen (70- and 80-kDa subunits) is a regulatory subunit of DNA-dependent protein kinase (DNA-PK) that promotes the recruitment of the catalytic subunit of DNA-PK (DNA-PKcs) to DNA ends and to specific DNA sequences from which the kinase is activated. Ku and DNA-PKcs plays essential roles in double-stranded DNA break repair and V(D)J recombination and have been implicated in the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a specific interaction between the 70-kDa subunit of Ku heterodimer and the homeodomain of HOXC4, a homeodomain protein expressed in the hematopoietic system. Unexpectedly, a similar interaction with Ku was observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of Ku70 and was abrogated by amino acid substitutions at Lys595/Lys596. Ku binding allowed the recruitment of the homeodomain to DNA ends and dramatically enhanced the phosphorylation of homeodomain-containing proteins by DNA-PK. These results suggest that Ku functions as a substrate docking protein for signaling by DNA-PK to homeodomain proteins from DNA ends.
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nuclear localization of Ku Antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits
Journal of Cell Science, 2001Co-Authors: Jesse Bertinato, Caroline Schildpoulter, Robert J. G. HachéAbstract:The Ku Antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein. Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase. While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood. In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently. A bipartite basic nuclear localization signal between amino acids 539–556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565–568 containing three lysines was required for the nuclear import of full-length Ku80. Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm. Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells.
Westley H Reeves - One of the best experts on this subject based on the ideXlab platform.
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Role of Antigen Selectivity in Autoimmune Responses to the Ku (p70/p80) Antigen
2016Co-Authors: Westley H Reeves, Z M Sthoeger, Robert G LahitaAbstract:Levels of anti-Ku (p7O/p8O) antibodies were measured longi-tudinally in sera from four individuals with systemic lupus erythematosus or related disorders. Antibodies to the native Ku Antigen (p7O/p8O complex) varied over a range of up to 577-fold. Large fluctuations were also observed in the levels of autoantibodies to several distinct epitopes of the Ku (p70/p8O) Antigen. Levels of these individual autoantibody populations generally paralleled one another, suggesting that they are coor-dinately regulated. A similar pattern of anti-DNA antibody fluctuation was seen in some sera. To examine the possibility that these autoantibodies were generated by polyclonal B cell activation, the levels ofanti-Ku (p70/p80) and anti-DNA anti-bodies were compared to the levels of antibodies to Escherichia coli proteins, tetanus toxoid, and bovine insulin, transferrin, cytochrome c, serum albumin, and thyroglobulin. In sera from the same individual, anti-Ku (p70/p80) antibodies were some-times produced in the complete absence of polyclonal activa-tion, and at other times were accompanied by increased poly-clonal activation. Anti-DNA antibody levels more closely par-alleled the level of polyclonal activation than did the anti-Ku (p70/p80) levels. These studies suggest that anti-Ku (p70/ p80) antibodies are generated by an Antigen-selective mecha-nism, but that polyclonal activation frequently, although not invariably, accompanies autoantibody production. This obser-vation is consistent with the possibility that polyclonal activa-tion might be secondary to autoantibody production
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a model for Ku heterodimer assembly and interaction with dna implications for the function of Ku Antigen
Journal of Biological Chemistry, 1998Co-Authors: Jingsong Wang, Xingwen Dong, Westley H ReevesAbstract:Ku autoAntigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371–510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179–510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179–732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases.
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human autoantibodies stabilize the quaternary structure of Ku Antigen
Arthritis & Rheumatism, 1997Co-Authors: Jingsong Wang, Minoru Satoh, Xingwen Dong, Lovorka Stojanov, Donald Kimpel, Westley H ReevesAbstract:Objective. To examine humoral immune responses to the native Ku Antigen and to evaluate the role of autoantibodies in stabilizing intermolecular contacts between the p70 and p80 Ku subunits. Methods. Recombinant free human p70 and p80 Ku subunits and p70/p80 heterodimers were expressed in Sf9 (insect) cells using baculovirus vectors. Affinity-purified recombinant human p70, p80, and p70/p80 dimer were studied by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation to evaluate autoantibody specificities in sera from 58 patients with systemic autoimmune disease. Results. Anti-Ku antibodies were detected by ELISA or immunoprecipitation using K562 cell Ku Antigen. All of the sera were reactive with the native recombinant p70, p80, or p70/p80 Antigens: 47% were anti-p70+,anti-p80+ and 32% were anti-p70-,anti-p80+, but only 3% were anti-p70+,anti-p80-. Unexpectedly, 18% of the sera recognized the p70/p80 dimer but did not recognize native p70 or p80 alone. A subset of sera containing autoantibodies that prevent the dissociation of p70 from p80 by high salt and detergent treatment was identified; monoclonal antibody (MAb) 162, a murine anti-Ku MAb, displays the same property. Autoantibodies that stabilize the p70-p80 interaction were found most frequently in sera containing both anti-p70 and anti-p80 antibodies. Conclusion. Autoantibodies to the native p80 sub-unit of Ku are more common than are anti-p70 antibodies. When anti-p70 antibodies were detected, they generally were found together with anti-p80. A novel type of autoantibody capable of stabilizing the p70/p80 heterodimer was identified in human sera for the first time. These “stabilizing” autoantibodies are found in sera containing both anti-p70 and anti-p80 antibodies, and also are produced by mice immunized with human Ku Antigen. Autoimmunity to Ku may be initiated with an immune response to p80, followed by spreading to p70. We hypothesize that stabilizing antibodies could facilitate the spreading of autoimmunity from one subunit of Ku to another by altering the processing of p70 or p80 by Antigen-presenting cells.
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role of free p70 Ku subunit in posttranslational stabilization of newly synthesized p80 during dna dependent protein kinase assembly
European Journal of Cell Biology, 1995Co-Authors: Minoru Satoh, Jingsong Wang, Westley H ReevesAbstract:The Ku Antigen (p70/p80 heterodimer) is the DNA binding component of a DNA-dependent serine/threonine kinase (DNA-PK), the catalytic activity of which is carried by a 350 kDa polypeptide (p350). In the present studies, the assembly of p70, p80, and p350 was investigated in human K562 (erythroleukemia) cells, and rabbit (RK13) or murine (L-929) cells infected with recombinant vaccinia viruses directing the synthesis of human p70 and p80. Pulse-chase analysis and density gradient centrifugation revealed a pool of free p70 subunits in K562 cells that dimerized within minutes with newly synthesized p80, whereas Ku became associated with newly synthesized p350 1 to 4 h after the onset of p70/p80 heterodimer assembly. A stable pool of free p80 subunits was not detected, and newly synthesized p80 was degraded rapidly (t1/2 16 h in RK13 cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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role of a major autoepitope in forming the dna binding site of the p70 Ku Antigen
Journal of Experimental Medicine, 1992Co-Authors: Chihhao Chou, Jingsong Wang, Mark W Knuth, Westley H ReevesAbstract:The Ku Antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.
