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Aprataxin

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Keith W Caldecott – 1st expert on this subject based on the ideXlab platform

  • versatility in phospho dependent molecular recognition of the xrcc1 and xrcc4 dna damage scaffolds by Aprataxin family fha domains
    DNA Repair, 2015
    Co-Authors: Amy L Cherry, Keith W Caldecott, Timothy J Nott, Geoffrey Kelly, Stuart L Rulten, Stephen J Smerdon

    Abstract:

    Aprataxin, Aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of Aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.

  • synergistic decrease of dna single strand break repair rates in mouse neural cells lacking both tdp1 and Aprataxin
    DNA Repair, 2009
    Co-Authors: Sherif F Elkhamisy, Sachin Katyal, Peter J Mckinnon, Limei Ju, Poorvi Patel, Keith W Caldecott

    Abstract:

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of Aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on Aprataxin activity by slowing the rate of repair of 3′-termini in Aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for Aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.

  • short patch single strand break repair in ataxia oculomotor apraxia 1
    Biochemical Society Transactions, 2009
    Co-Authors: John J Reynolds, Sherif F Elkhamisy, Keith W Caldecott

    Abstract:

    AOA1 (ataxia oculomotor apraxia-1) results from mutations in Aprataxin, a component of DNA strand break repair that removes AMP from 5-termini. In the present article, we provide an overview of this disease and review recent experiments demonstrating that short-patch repair of oxidative single-strand breaks in AOA1 cell extracts bypasses the point of Aprataxin action and stalls at the final step of DNA ligation, resulting in accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of non-adenylated DNA ligase and short-patch single-strand break repair can be restored in AOA1 extracts, independently of Aprataxin, by addition of recombinant DNA ligase.

Sherif F Elkhamisy – 2nd expert on this subject based on the ideXlab platform

  • synergistic decrease of dna single strand break repair rates in mouse neural cells lacking both tdp1 and Aprataxin
    DNA Repair, 2009
    Co-Authors: Sherif F Elkhamisy, Sachin Katyal, Peter J Mckinnon, Limei Ju, Poorvi Patel, Keith W Caldecott

    Abstract:

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of Aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on Aprataxin activity by slowing the rate of repair of 3′-termini in Aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for Aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1.

  • short patch single strand break repair in ataxia oculomotor apraxia 1
    Biochemical Society Transactions, 2009
    Co-Authors: John J Reynolds, Sherif F Elkhamisy, Keith W Caldecott

    Abstract:

    AOA1 (ataxia oculomotor apraxia-1) results from mutations in Aprataxin, a component of DNA strand break repair that removes AMP from 5-termini. In the present article, we provide an overview of this disease and review recent experiments demonstrating that short-patch repair of oxidative single-strand breaks in AOA1 cell extracts bypasses the point of Aprataxin action and stalls at the final step of DNA ligation, resulting in accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of non-adenylated DNA ligase and short-patch single-strand break repair can be restored in AOA1 extracts, independently of Aprataxin, by addition of recombinant DNA ligase.

  • defective dna ligation during short patch single strand break repair in ataxia oculomotor apraxia 1
    Molecular and Cellular Biology, 2009
    Co-Authors: John J Reynolds, Sherif F Elkhamisy, Sachin Katyal, Paula M Clements, Peter J Mckinnon, Keith W Caldecott

    Abstract:

    Ataxia oculomotor apraxia 1 (AOA1) results from mutations in Aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other Aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of Aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of Aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in Aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx(-/-) neural astrocytes. These data demonstrate that Aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase.

Martin F Lavin – 3rd expert on this subject based on the ideXlab platform

  • Aprataxin poly adp ribose polymerase 1 parp 1 and apurinic endonuclease 1 ape1 function together to protect the genome against oxidative damage
    Human Molecular Genetics, 2009
    Co-Authors: Olivier J Becherel, Martin F Lavin, Burkhard Jakob, Gisela Taucherscholz, Janelle L Harris, Grigory L Dianov

    Abstract:

    Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1 (AOA1), is a DNA repair protein that processes the product of abortive ligations, 5′ adenylated DNA. In addition to its interaction with the single-strand break repair protein XRCC1, Aprataxin also interacts with poly-ADP ribose polymerase 1 (PARP-1), a key player in the detection of DNA single-strand breaks. Here, we reveal reduced expression of PARP-1, apurinic endonuclease 1 (APE1) and OGG1 in AOA1 cells and demonstrate a requirement for PARP-1 in the recruitment of Aprataxin to sites of DNA breaks. While inhibition of PARP activity did not affect Aprataxin activity in vitro, it retarded its recruitment to sites of DNA damage in vivo. We also demonstrate the presence of elevated levels of oxidative DNA damage in AOA1 cells coupled with reduced base excision and gap filling repair efficiencies indicative of a synergy between Aprataxin, PARP-1, APE-1 and OGG1 in the DNA damage response. These data support both direct and indirect modulating functions for Aprataxin on base excision repair.

  • nucleolar localization of Aprataxin is dependent on interaction with nucleolin and on active ribosomal dna transcription
    Human Molecular Genetics, 2006
    Co-Authors: Olivier J Becherel, Martin F Lavin, Nuri Gueven, Geoff W Birrell, Valeerie Schreiber, Amila Suraweera, Burkhard Jakob, Gisela Taucherscholz

    Abstract:

    The APTX gene, mutated in patients with the neurological disorder ataxia with oculomotor apraxia type 1 (AOA1), encodes a novel protein Aprataxin. We describe here, the interaction and interdependence between Aprataxin and several nucleolar proteins, including nucleolin, nucleophosmin and upstream binding factor-1 (UBF-1), involved in ribosomal RNA (rRNA) synthesis and cellular stress signalling. Interaction between Aprataxin and nucleolin occurred through their respective N-terminal regions. In AOA1 cells lacking Aprataxin, the stability of nucleolin was significantly reduced. On the other hand, down-regulation of nucleolin by RNA interference did not affect Aprataxin protein levels but abolished its nucleolar localization suggesting that the interaction with nucleolin is involved in its nucleolar targeting. GFP-Aprataxin fusion protein co-localized with nucleolin, nucleophosmin and UBF-1 in nucleoli and inhibition of ribosomal DNA transcription altered the distribution of Aprataxin in the nucleolus, suggesting that the nature of the nucleolar localization of Aprataxin is also dependent on ongoing rRNA synthesis. In vivo rRNA synthesis analysis showed only a minor decrease in AOA1 cells when compared with controls cells. These results demonstrate a cross-dependence between Aprataxin and nucleolin in the nucleolus and while Aprataxin does not appear to be directly involved in rRNA synthesis its nucleolar localization is dependent on this synthesis.

  • Aprataxin forms a discrete branch in the hit histidine triad superfamily of proteins with both dna rna binding and nucleotide hydrolase activities
    Journal of Biological Chemistry, 2006
    Co-Authors: Amanda W Kijas, Janelle L Harris, Jonathan M Harris, Martin F Lavin

    Abstract:

    Abstract Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset autosomal recessive spinocerebellar ataxia with a defect in the protein Aprataxin, implicated in the response of cells to DNA damage. We describe here the expression of a recombinant form of Aprataxin and show that it has dual DNA binding and nucleotide hydrolase activities. This protein binds to double-stranded DNA with high affinity but is also capable of binding double-stranded RNA and single-strand DNA, with increased affinity for hairpin structures. No increased binding was observed with a variety of DNA structures mimicking intermediates in DNA repair. The DNA binding observed here was not dependent on zinc, and the addition of exogenous zinc abolished DNA binding. We also demonstrate that Aprataxin hydrolyzes with similar efficiency the model histidine triad nucleotide-binding protein substrate, AMPNH2, and the Fragile histidine triad protein substrate, Ap4A. These activities were significantly reduced in the presence of duplex DNA and to a lesser extent in the presence of single-strand DNA, and removal of the N-terminal Forkhead associated domain did not alter activity. Finally, comparison of sequence relationships between the histidine triad superfamily members shows that Aprataxin forms a distinct branch in this superfamily. In addition to its capacity for nucleotide binding and hydrolysis, the observation that it also binds DNA and RNA adds a new dimension to this superfamily of proteins and provides further support for a role for Aprataxin in the cellular response to DNA damage.