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.
Article: PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation.[show abstract] [hide abstract]
ABSTRACT: In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase delta-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase delta and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.Cell cycle (Georgetown, Tex.) 11/2008; 7(20):3180-6. · 5.36 Impact Factor
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ABSTRACT: Using a yeast two-hybrid screening technique and the p50 subunit of human DNA polymerase delta (pol delta) as a bait, p21 was found to interact with the p50 subunit of pol delta. A direct interaction between p21 and p50 was confirmed by using ELISA and pull-down assays with purified proteins. The interaction sites between p50 and p21 were mapped by pull down assays with GST deletion mutants. Residues 127-193 constitute the primary interaction region on p50 to which p21 binds, while p50 binds to the C-terminal 26 residues of p21. A histone kinase activity was associated with the highly purified calf thymus pol delta and addition of purified recombinant p21 inhibited the kinase activity in a dose dependent manner. p50 is phosphorylated in vivo and can be phosphorylated by CDK2/cyclinA in vitro. In vivo evidence of p21 association with p50 was obtained by coimmunoprecipitation using MCF7 cells. It was also shown that the association of p21 with p50 and other components of the pol delta complex increased in MCF7 cells treated with adriamycin. Our results suggested that p50 might target or anchor p21 to pol delta complex upon certain DNA damage such as adriamycin treatment.Cell cycle (Georgetown, Tex.) 03/2006; 5(4):428-36. · 5.36 Impact Factor
Article: SEXUAL DIMORPHISM OF THYROID REACTIVE OXYGEN SPECIES PRODUCTION DUE TO HIGHER NOX4 EXPRESSION IN FEMALE THYROIDS.[show abstract] [hide abstract]
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