RESUMEN
The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.
Asunto(s)
Inestabilidad Cromosómica , ADN Helicasas/metabolismo , Replicación del ADN , Animales , Supervivencia Celular , ADN Helicasas/química , ADN Mitocondrial/metabolismo , Enfermedad/genética , HumanosRESUMEN
The disease-associated nuclease-helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.
Asunto(s)
ADN Helicasas/genética , Reparación del ADN/genética , Replicación del ADN/genética , Recombinación Homóloga/genética , Supervivencia Celular/genética , ADN/genética , Enanismo/genética , Genoma Humano/genética , Humanos , Neoplasias/genéticaRESUMEN
DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Enfermedades Genéticas Congénitas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN , Daño del ADN , ADN Helicasas/genética , Humanos , Mutación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA double-strand breaks (DSBs) disrupt the structural integrity of chromosomes. Proper DSB repair pathway choice is critical to avoid the type of gross chromosomal rearrangements that characterize cancer cells. Recent findings reveal S-fatty acylation and membrane anchorage of Rap1-interacting factor 1 (Rif1) as a mechanism providing spatial control over DSB repair pathway choice.
RESUMEN
Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión a Telómeros/genética , Acilación , Reparación del ADN , Membrana Nuclear/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.
Asunto(s)
Cromosomas Humanos/genética , Replicación del ADN , Endonucleasas/genética , Neoplasias/genética , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Resolvasas de Unión Holliday/genética , Humanos , Recombinasas/genética , Estrés FisiológicoRESUMEN
Cells have evolved conserved mechanisms to protect DNA ends, such as those at the termini of linear chromosomes, or those at DNA double-strand breaks (DSBs). In eukaryotes, DNA ends at chromosomal termini are packaged into proteinaceous structures called telomeres. Telomeres protect chromosome ends from erosion, inadvertent activation of the cellular DNA damage response (DDR), and telomere fusion. In contrast, cells must respond to damage-induced DNA ends at DSBs by harnessing the DDR to restore chromosome integrity, avoiding genome instability and disease. Intriguingly, Rif1 (Rap1-interacting factor 1) has been implicated in telomere homeostasis as well as DSB repair. The protein was first identified in Saccharomyces cerevisiae as being part of the proteinaceous telosome. In mammals, RIF1 is not associated with intact telomeres, but was found at chromosome breaks, where RIF1 has emerged as a key mediator of pathway choice between the two evolutionary conserved DSB repair pathways of non-homologous end-joining (NHEJ) and homologous recombination (HR). While this functional dichotomy has long been a puzzle, recent findings link yeast Rif1 not only to telomeres, but also to DSB repair, and mechanistic parallels likely exist. In this review, we will provide an overview of the actions of Rif1 at DNA ends and explore how exclusion of end-processing factors might be the underlying principle allowing Rif1 to fulfill diverse biological roles at telomeres and chromosome breaks.
RESUMEN
In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved â¼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/enzimologíaRESUMEN
DNA replication is mediated by a multi-protein complex known as the replisome. With the hexameric MCM (minichromosome maintenance) replicative helicase at its core, the replisome splits the parental DNA strands, forming replication forks (RFs), where it catalyses coupled leading and lagging strand DNA synthesis. While replication is a highly effective process, intrinsic and oncogene-induced replication stress impedes the progression of replisomes along chromosomes. As a consequence, RFs stall, arrest, and collapse, jeopardizing genome stability. In these instances, accessory fork progression and repair factors, orchestrated by the replication checkpoint, promote RF recovery, ensuring the chromosomes are fully replicated and can be safely segregated at cell division. Homologous recombination (HR) proteins play key roles in negotiating replication stress, binding at stalled RFs and shielding them from inappropriate processing. In addition, HR-mediated strand exchange reactions restart stalled or collapsed RFs and mediate error-free post-replicative repair. DNA transactions at stalled RFs further involve various DNA editing factors, notably helicases and nucleases. A study by Ölmezer et al. (2016) has recently identified a role for the structure-specific nuclease Yen1 (GEN1 in human) in the resolution of dead-end DNA replication intermediates after RF arrest. This new function of Yen1 is distinct from its previously known role as a Holliday junction resolvase, mediating the removal of branched HR intermediates, and it becomes essential for viable chromosome segregation in cells with a defective Dna2 helicase. These findings have revealed greater complexity in the tasks mediated by Yen1 and expose a replicative role for the elusive helicase activity of the conserved Dna2 nuclease-helicase.
RESUMEN
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN , Regulación Fúngica de la Expresión Génica , Resolvasas de Unión Holliday/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Segregación Cromosómica , Cromosomas Fúngicos/química , Cromosomas Fúngicos/metabolismo , ADN Helicasas/metabolismo , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Resolvasas de Unión Holliday/metabolismo , Recombinación Homóloga , Mitosis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
PBY1 continues to be linked with DNA repair through functional genomics studies in yeast. Using the yeast knockout (YKO) strain collection, high-throughput genetic interaction screens have identified a large set of negative interactions between PBY1 and genes involved in genome stability. In drug sensitivity screens, the YKO collection pby1Δ strain exhibits a sensitivity profile typical for genes involved in DNA replication and repair. We show that these findings are not related to loss of Pby1. On the basis of genetic interaction profile similarity, we pinpoint disruption of Holliday junction resolvase Mus81-Mms4 as the mutation responsible for DNA repair phenotypes currently ascribed to pby1. The finding that Pby1 is not a DNA repair factor reconciles discrepancies in the data available for PBY1, and indirectly supports a role for Pby1 in mRNA metabolism. Data that has been collected using the YKO collection pby1Δ strain confirms and expands the chemical-genetic interactome of MUS81-MMS4.
Asunto(s)
Reparación del ADN , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Cromosomas Fúngicos/metabolismo , Daño del ADN , Epistasis Genética , Técnicas de Inactivación de Genes , Mutación/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81-Mms4/MUS81-EME1, Yen1/GEN1, and Slx1-Slx4/SLX1-SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process.
Asunto(s)
Replicación del ADN , ADN/química , Endonucleasas/genética , Eucariontes/genética , Animales , ADN/genética , Reparación del ADN , Endonucleasas/metabolismo , Eucariontes/metabolismo , Genoma , Resolvasas de Unión Holliday , Recombinación Homóloga , Humanos , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Yeast telomeres comprise irregular TG1â3 DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition of telomeres as DNA double-strand breaks. We provide a molecular, biochemical, and functional dissection of the protein backbone at the core of the yeast telosome. The X-ray structures of Rif1 and Rif2 bound to the Rap1 C-terminal domain and that of the Rif1 C terminus are presented. Both Rif1 and Rif2 have separable and independent Rap1-binding epitopes, allowing Rap1 binding over large distances (42-110 Å). We identify tetramerization (Rif1) and polymerization (Rif2) modules that, in conjunction with the long-range binding, give rise to a higher-order architecture that interlinks Rap1 units. This molecular Velcro relies on Rif1 and Rif2 to recruit and stabilize Rap1 on telomeric arrays and is required for telomere homeostasis in vivo.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Mapas de Interacción de Proteínas , Alineación de Secuencia , Complejo ShelterinaRESUMEN
Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1-HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5'-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.
Asunto(s)
ADN Cruciforme/metabolismo , Resolvasas de Unión Holliday/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Resolvasas de Unión Holliday/química , Humanos , Especificidad por SustratoRESUMEN
In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Delta mutants are repair-proficient, yen1Delta mus81Delta double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Delta mus81Delta double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Resolvasas de Unión Holliday/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Roturas del ADN de Doble Cadena , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endonucleasas de ADN Solapado/genética , Resolvasas de Unión Holliday/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Four-way DNA intermediates, also known as Holliday junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday junction resolvase RuvC. The human Holliday junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. The eukaryotic Holliday junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday junctions by the introduction of symmetrically related cuts across the junction point, to produce nicked duplex products in which the nicks can be readily ligated.
Asunto(s)
Resolvasas de Unión Holliday/aislamiento & purificación , Resolvasas de Unión Holliday/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN/química , ADN/metabolismo , Reparación del ADN , Células HeLa , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/genética , Humanos , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
The human neurological disease known as ataxia with oculomotor apraxia 1 is caused by mutations in the APTX gene that encodes Aprataxin (APTX) protein. APTX is a member of the histidine triad superfamily of nucleotide hydrolases and transferases but is distinct from other family members in that it acts upon DNA. The target of APTX is 5'-adenylates at DNA nicks or breaks that result from abortive DNA ligation reactions. In this work, we show that APTX acts as a nick sensor, which provides a mechanism to assess the adenylation status of unsealed nicks. When an adenylated nick is encountered by APTX, base pairing at the 5' terminus of the nick is disrupted as the adenylate is accepted into the active site of the enzyme. Adenylate removal occurs by a two-step process that proceeds through a transient AMP-APTX covalent intermediate. These results pinpoint APTX as the first protein to adopt canonical histidine triad-type reaction chemistry for the repair of DNA.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , ADN/química , Enfermedades del Sistema Nervioso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Animales , Secuencia de Bases , Línea Celular , Pollos , ADN Complementario/metabolismo , Desoxirribonucleasa I/química , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Defects in cellular DNA repair processes have been linked to genome instability, heritable cancers, and premature aging syndromes. Yet defects in some repair processes manifest themselves primarily in neuronal tissues. This review focuses on studies defining the molecular defects associated with several human neurological disorders, particularly ataxia with oculomotor apraxia 1 (AOA1) and spinocerebellar ataxia with axonal neuropathy 1 (SCAN1). A picture is emerging to suggest that brain cells, due to their nonproliferative nature, may be particularly prone to the progressive accumulation of unrepaired DNA lesions.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Apraxias/genética , Apraxias/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Axones/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Humanos , Modelos Moleculares , Mutación , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Proteínas Nucleares/química , Proteínas Nucleares/genética , Enfermedades del Nervio Oculomotor/genética , Enfermedades del Nervio Oculomotor/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Conformación Proteica , Estructura Terciaria de Proteína , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/metabolismo , Dedos de ZincRESUMEN
Mutations in the Aptx gene lead to a neurological disorder known as ataxia oculomotor apraxia-1. The product of Aptx is Aprataxin (Aptx), a DNA-binding protein that resolves abortive DNA ligation intermediates. Aprataxin catalyzes the nucleophilic release of adenylate groups covalently linked to 5' phosphate termini, resulting in termini that can again serve as substrates for DNA ligases. Here we show that Aprataxin acts preferentially on adenylated nicks and double-strand breaks rather than on single-stranded DNA. Moreover, we show that whereas the catalytic activity of Aptx resides within the HIT domain, the C-terminal zinc finger domain provides stabilizing contacts that lock the enzyme onto its high affinity AMP-DNA target site. Both domains are therefore required for efficient AMP-DNA hydrolase activity. Additionally, we find a role for Aprataxin in base excision repair, specifically in the removal of adenylates that arise from abortive ligation reactions that take place at incised abasic sites in DNA. We suggest that Aprataxin may have a general proofreading function in DNA repair, removing DNA adenylates as they arise during single-strand break repair, double-strand break repair, and in base excision repair.