Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive form of cerebellar ataxia. The causative protein for EAOH/AOA1, aprataxin (APTX), interacts with X-ray repair cross-complementing 1 (XRCC1), a scaffold DNA repair protein for single-strand breaks (SSBs). The goal of this study was to prove the functional involvement of APTX in SSB repair (SSBR).
We visualized the SSBR process with a recently developed laser irradiation system that allows real-time observation of SSBR proteins and with a local ultraviolet-irradiation system using a XPA-UVDE cell line that repairs DNA lesions exclusively via SSBR. APTX was knocked down using small interference RNA in the cells. Oxidative stress-induced DNA damage and cell death were assessed in EAOH fibroblasts and cerebellum.
Our systems showed the XRCC1-dependent recruitment of APTX to SSBs. SSBR was impaired in APTX-knocked-down cells. Oxidative stress in EAOH fibroblasts readily induced SSBs and cell death, which were blocked by antioxidants. Accumulated oxidative DNA damage was confirmed in EAOH cerebellum.
This study provides the first direct evidence for the functional involvement of APTX in SSBR and in vivo DNA damage in EAOH/AOA1, and suggests a benefit of antioxidant treatment.
"The characteristic shared by all these disorders is a high level of spontaneously occurring chromosome abnormalities as well as the hypersensitivity to chromosome aberrations induced by genotoxic agents. In addition, there are other disorders caused by mutations in genes involved in DNA SSB repair, such as ataxiaoculomotor apraxia, in which cells from patients are hypersensitive to genotoxic agents inducing SSB  . All these disorders are autosomal recessive and the hypersensitivity to genotoxic agents is demonstrated in cells originating from homozygous subjects; however, an increased hypersensitivity has also been detected in cells from heterozygous subjects . "
[Show abstract][Hide abstract] ABSTRACT: In addition to the main function of promoting polymerization and stabilization of microtubules, other roles are being attributed to tau, now considered a multifunctional protein. In particular, previous studies suggest that tau is involved in chromosome stability and genome protection. We performed cytogenetic analysis, including molecular karyotyping, on lymphocytes and fibroblasts from patients affected by frontotemporal lobar degeneration carrying different mutations in the microtubule-associated protein tau gene, to investigate the effects of these mutations on genome stability. Furthermore, we analyzed the response of mutated lymphoblastoid cell lines to genotoxic agents to evaluate the participation of tau to DNA repair systems. We found a significantly higher level of chromosome aberrations in mutated than in control cells. Mutated lymphocytes showed higher percentages of stable lesions, clonal and total aneuploidy (medians: 2 versus 0, p $\ll$ 0.01; 1.5 versus 0, p $\ll$ 0.01; 16.5 versus 0, p $\ll$ 0.01, respectively). Fibroblasts of patients showed higher percentages of stable lesions, structural aberrations and total aneuploidy (medians: 0 versus 0, p = 0.03; 5.8 versus 0, p = 0.02; 26.5 versus 12.6, p $\ll$ 0.01, respectively). In addition, the in depth analysis of DNA copy number variations showed a higher tendency to non-allelic homologous recombination in mutated cells. Finally, while our analysis did not support an involvement of tau in DNA repair systems, it revealed its role in stabilization of chromatin. In summary, our findings indicate a role of tau in genome and chromosome stability that can be ascribed to its function as a microtubule-associated protein as well as a protein protecting chromatin integrity through interaction with DNA.
"Cells from AOA1 patients are also not particularly susceptible to genotoxic agents, including methyl methane sulphonate (MMS) and hydrogen peroxide (Clements et al., 2004; El-Khamisy et al., 2009). Indeed, the main function of aprataxin may not reside in the nucleus, as APTX knockout mice do not exhibit a clear defect in SSBR and aprataxin does not appear to re-localize to nuclear DNA lesions after exposure to a range of DNA damaging agents (Hirano et al., 2007). These observations led us to hypothesize that AOA1 may stem from mitochondrial dysfunction, rather than nuclear repair defect. "
[Show abstract][Hide abstract] ABSTRACT: Oxidative DNA damage has been attributed to increased cancer incidence and premature aging phenotypes. Reactive oxygen species (ROS) are unavoidable byproducts of oxidative phosphorylation and are the major contributors of endogenous oxidative damage. To prevent the negative effects of ROS, cells have developed DNA repair mechanisms designed to specifically combat endogenous DNA modifications. The base excision repair (BER) pathway is primarily responsible for the repair of small non-helix distorting lesions and DNA single strand breaks. This repair pathway is found in all organisms, and in mammalian cells, consists of three related sub-pathways: short patch (SP-BER), long patch (LP-BER) and single strand break repair (SSBR). While much is known about nuclear BER, comparatively little is known about this pathway in the mitochondria, particularly the LP-BER and SSBR sub-pathways. There are a number of proteins that have recently been found to be involved in mitochondrial BER, including Cockayne syndrome proteins A and B (CSA and CSB), aprataxin (APTX), tryosyl-DNA phosphodiesterase 1 (TDP1), flap endonuclease 1 (FEN-1) and exonuclease G (EXOG). These significant advances in mitochondrial DNA repair may open new avenues in the management and treatment of a number of neurological disorders associated with mitochondrial dysfunction, and will be reviewed in further detail herein.
Mechanisms of ageing and development 11/2011; 133(4):169-75. DOI:10.1016/j.mad.2011.11.003 · 3.40 Impact Factor
"Environmental and Molecular Mutagenesis 52:623^635 (2011) 2005; Hirano et al., 2007]. Xrcc1 2/2 cells were recently shown to have a slightly reduced rate of repair of uracil in DNA, but not repair of AP sites [Akbari et al., 2010]. "
[Show abstract][Hide abstract] ABSTRACT: XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLβ and PNK colocalize with XRCC1 in replication foci and that POLβ and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLβ and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLβ to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.
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