The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

Abstract

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Neil2-null Mice Accumulate Oxidized DNA Bases in the Transcriptionally Active Sequences of the Genome and Are Susceptible to Innate Inflammation

“…LA-qPCR for DNA Damage Analysis-Long amplicon quantitative-PCR (LA-qPCR) assays were carried out essentially following the reported protocols (19,(23)(24)(25) with some minor modifications. Briefly, livers (15 mg), kidneys (20 mg), lungs (20 mg), and whole brain (15 mg) were harvested from WT and…”

“…LA-qPCR was carried out to amplify a 6.5-kb region of pol ␤, 8.7 kb of ␤-globin, 7.2 kb of NeuroD, and 7.4 kb of NanoG in mouse genomic DNA using the primer sets described in Table 1. The numbers of cycles and DNA concentrations were standardized in each case before the actual reaction, so that the PCR remains within the linear range of amplification (23)(24)(25). The final PCR condition was optimized at 94°C for 30 s (94°C for 30 s, 55-60°C for 30 s depending on the oligo annealing temperature, 65°C for 10 min) for 25 cycles and 65°C for 10 min.…”

Base Excision Repair, a Pathway Regulated by Posttranslational Modifications

“…Therefore, cross talk between phosphorylation and ubiquitylation plays a critical role in the regulation of PNKP. Interestingly, PNKP has recently been shown to interact in vivo with the deubiquitylation enzyme ataxin-3 (ATXN3), which enhances PNKP phosphatase activity (73). Therefore, it is possible that deubiquitylation of PNKP by ATXN3 is opposing Cul4A-DDB1-STRAP-mediated ubiquitylation of PNKP.…”

Ataxia espinocerebelosa tipo 3 (enfermedad de Machado-Joseph) y vareniclina

“…Nuevos hallazgos han señalado que la mutación del gen ATXN3 (ubicado en el brazo largo del cromosoma 14) produce un incremento de las expansiones CAG, induciendo acumulación de residuos largos de glutamina o poliglutamina (y toxicidad por proteínas), lo que resulta en la enfermedad 7 . Además, se observó que las repeticiones de la secuencia CAG del gen ATXN3 inhiben la actividad de la polinucleótido quinasa 3 fosfatasa (PNKP) con la consiguiente acumulación de ADN dañado 8 . Se ha observado una disminución de la actividad enzimática en modelos animales sobre las regiones de sistema nervioso afectadas por la AET3 y en cerebros de pacientes con AET3, dado que existe una acumulación significativa del ADN dañado.…”

“…Se ha observado una disminución de la actividad enzimática en modelos animales sobre las regiones de sistema nervioso afectadas por la AET3 y en cerebros de pacientes con AET3, dado que existe una acumulación significativa del ADN dañado. Se ha demostrado que la acumulación del ADN dañado indujo muerte neuronal, lo que es un hallazgo común en estos pacientes 8 . Será importante determinar si la normalización de los niveles y/o actividad de ésta enzima beneficiaría a estos pacientes.…”