Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen.
ABSTRACT The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta). Two baits were used in this study. These were the large (p125) and small (p50) subunits of the core pol delta heterodimer. p50 was the only positive isolated with p125 as the bait. Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen. In this study, the interaction of PDIP38 with pol delta was further characterized. PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein. It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA). The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins. The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments. The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments. PDIP38 was associated with pol delta isolated by immunoaffinity chromatography. The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis.
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ABSTRACT: TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5'-GA-3' and 5'-UG-3' repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splice-dependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.Nucleic Acids Research 11/2011; 40(6):2668-82. · 8.28 Impact Factor
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ABSTRACT: The p12 subunit of polymerase delta (Pol δ) is degraded in response to DNA damage induced by UV, alkylating agents, oxidative, and replication stresses. This leads to the conversion of the Pol δ4 holoenzyme to the heterotrimer, Pol δ3. We review studies that establish that Pol δ3 formation is an event that could have a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. p12 degradation is dependent on the apical ataxia telangiectasia and Rad3 related (ATR) kinase and is mediated by the ubiquitin-proteasome system. Pol δ3 exhibits properties of an "antimutator" polymerase, suggesting that it could contribute to an increased surveillance against mutagenesis, for example, when Pol δ carries out bypass synthesis past small base lesions that engage in spurious base pairing. Chromatin immunoprecipitation analysis and examination of the spatiotemporal recruitment of Pol δ to sites of DNA damage show that Pol δ3 is the primary form of Pol δ associated with cyclobutane pyrimidine dimer lesions and therefore should be considered as the operative form of Pol δ engaged in DNA repair. We propose a model for the switching of Pol δ with translesion polymerases, incorporating the salient features of the recently determined structure of monoubiquitinated proliferating cell nuclear antigen and emphasizing the role of Pol δ3. Because of the critical role of Pol δ activity in DNA replication and repair, the formation of Pol δ3 in response to DNA damage opens the prospect that pleiotropic effects may ensue. This opens the horizons for future exploration of how this novel response to DNA damage contributes to genomic stability. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.Environmental and Molecular Mutagenesis 10/2012; · 3.71 Impact Factor
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ABSTRACT: Background: Dual oxidases (DUOX1 and DUOX2) are NADPH oxidases involved in hydrogen peroxide production necessary for thyroid hormonogenesis, but recently the NADPH oxidase 4 (NOX4) has also been described in the thyroid gland. The prevalence of thyroid disease is higher in women, and the basis for this difference might involve a higher oxidative stress level in the female thyroid gland. Hence, we aimed at evaluating whether the function and the expression of enzymes involved in thyroid redox balance differ between females and males. Methods: DUOX1 and 2, NOX4, glutathione peroxidase (GPx), and catalase activities and expression levels were evaluated in the thyroids of prebubertal and adult male and female rats. The mRNA levels of DUOXA1 and DUOXA2, the DUOX maturation factors, and of p22phox and Poldip2, subunits of NOX4, were also determined. Results: A higher calcium-independent H2O2 production was detected in the adult female rat thyroid, being higher in the estrous phase of the cycle. Moreover, the expression of NOX4 and Poldip2 mRNA was higher in the thyroid from adult female rats, as well as in PCCL3 cells treated with 17ß-estradiol. The GPx1 mRNA expression was higher in adult female thyroids, while GPx2 and GPx3 mRNA and total GPx activity were not significantly different. Catalase mRNA expression and activity, together with thyroid thiol levels were significantly lower in the adult female rat thyroid. Conclusions: Taken together, our results show that the thyroid gland of female rats is exposed to higher oxidative stress levels due to both increased ROS production through NOX4 and decreased ROS degradation.Thyroid: official journal of the American Thyroid Association 10/2012; · 2.60 Impact Factor