Stimulation of DNA Polymerase γ Activity by Proliferating Cell Nuclear Antigen

doi:10.1006/bbrc.1995.2680

Biochemical and Biophysical Research Communications

Volume 216, Issue 2, 13 November 1995, Pages 715-722

https://doi.org/10.1006/bbrc.1995.2680 Get rights and content

Abstract

DNA polymerase was partially purified from mitochondrial extracts of rat liver by phosphocellulose, DEAE-cellulose, heparin-Sepharose CL-6B and DNA-agarose column chromatography. By these purification steps, DNA polymerase and proliferating cell nuclear antigen (PCNA) were completely separated at the step of heparin-Sepharose CL-6B column chromatography. The isolated DNA polymerase was inhibited by ddTTP, but not by aphidicolin. The enzyme sedimented at about 8 S on 5-20 % analytical sucrose density gradient centrifugation. These data showed that the DNA polymerase isolated from mitochondria is γ in type. After the separation of DNA polymerase γ and PCNA, the two fractions were remixed and DNA polymerase γ activity was measured. DNA polymerase γ activity was stimulated about three-fold or more in the presence of the PCNA fraction. This stimulation was inhibited by the addition of anti-PCNA rabbit IgG2a. In addition, highly purified human recombinant PCNA stimulated the DNA polymerase γ activity. These results indicate that DNA polymerase γ, like DNA polymerase δ, is activated by PCNA.

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Cited by (6)

Further characterization of human DNA polymerase δ interacting protein 38
2005, Journal of Biological Chemistry
Citation Excerpt :
PCNA, which is generally considered to be a nuclear protein and whose functions are in the nucleus, has been reported to be able to stimulate mitochondrial polymerase γ activity (45). A more recent study (46) showed that monoclonal antibodies against PCNA specifically recognized a single band of 30 kDa in the mitochondria from the filamentous fungus and yeast. These studies suggest that PDIP38 might function in the mitochondria through its interaction with PCNA, even though the level of PCNA in mammalian mitochondria might be too low to be detected by Western blotting.
Polymerase δ interacting protein 38 (PDIP38) was identified as a human DNA polymerase (pol) δ interacting protein through a direct interaction with p50, the small subunit of human pol δ. PDIP38 was also found to interact with proliferating cell nuclear antigen, which suggested that it might play a role in vivo in the processes of DNA replication and DNA repair in the nucleus. In order to characterize further this novel protein, we have examined its subcellular localization by the use of immunochemical and cellular fractionation techniques. These studies show that PDIP38 is a novel mitochondrial protein and is localized mainly to the mitochondria. PDIP38 was shown to possess a functional mitochondrial targeting sequence that is located within the first 35 N-terminal amino acid residues. The mature PDIP38 protein is about 50 amino acid residues smaller than the full-length precursor PDIP38 protein, consistent with it being processed by cleavage of the mitochondrial targeting sequence during entry into the mitochondria. His-tagged mature PDIP38 inhibited pol δ activity in vitro and interacted with human papillomavirus 16 E7 oncoprotein, suggesting that PDIP38 might play a role in the pol δ-mediated viral DNA replication. Although the localization of PDIP38 to the mitochondria suggests that it serves functions within the mitochondria, we cannot eliminate the possibility that it may be involved in pol δ-mediated DNA replication or DNA repair under certain conditions such as viral infection.
Stimulation of a mitochondrial endo-exonuclease from Podospora anserina by PCNA
2003, Biochemical and Biophysical Research Communications
Citation Excerpt :
This size corresponds to the yeast PCNA pol30 gene product molecular weight of 28.4 kDa and to the fungal PCNA homolog, and thus further suggests the recognition specificity of this protein inside this organelle. In accordance with the detection in fungal mitochondria of a 30-kDa protein specifically recognized by anti-PCNA antibodies and the stimulation of the nuclease activity by the PCNA, a recent report described the 2–3-fold stimulation of the DNA polymerase γ activity from rat liver mitochondria and concomitantly the co-purification of immunostained PCNA revealed by immunoblotting in the polymerase γ fraction prepared from mt extracts [22]. We observed the same stimulation of mt DNA polymerase γ activity from P. anserina and S. cerevisiae (data not shown).
In Podospora anserina we have described the purification of an endo–exonuclease (Biochim. Biophys. Acta 1574 (1) (2002) 72). Given the description of several nucleases addressed to the mitochondria and known to interact with the PCNA, we sought a possible effect of PCNA on the mt nuclease. A significant stimulation of the nuclease activity with PCNA was observed with double-stranded and flap structure DNA. Immuno-Western blotting experiments realized with monoclonal antibodies raised against the PCNA specifically revealed the presence of a single band of 30 kDa in the mitochondria from the filamentous fungus and yeast. A potential PCNA binding motif was found in the sequences of several mt nucleases and mt proteins involved in the maintenance of the mt DNA with respect to the consensus described by Warbrick. The hypothetical role of the PCNA as a potential regulator of the repair/recombination processes in the maintenance of the mt genome is discussed.
Increase in DNA Polymerase γ in the Hearts of Adriamycin- Administered Rats
2002, Experimental and Molecular Pathology
It is hypothesized that the cause of myocardiopathy is oxidative damage to mitochondrial DNA. To clarify this hypothesis, DNA polymerase γ activity, which is related to the final step of mitochondrial DNA repair or renewal, was measured. One cycle of treatment consisted of five injections of adriamycin over 5 days at a dose of 1 mg/kg of body weight per day and then 2 days resting time. DNA polymerase γ activities in the heart after one cycle of treatment were lower than the control level. However, DNA polymerase γ activities increased with continued adriamycin treatment, reaching a maximum level in the heart at 14 days after two cycles of adriamycin treatment. Induction of DNA polymerase γ activity was found in rat heart following three and four cycles of administration. Under these conditions, it is doubtful that mitochondrial DNA is the direct target of adriamycin administration. The damaged mitochondrial DNA may be protected by actions of the renewal or repair systems, maintaining mitochondrial function in the heart. Rat hearts at 7 days after one cycle of adriamycin treatment show morphological changes in the mitochondria that include matrix swelling and cristae disorganization, as seen in cardiac cells by electron microscopy; however, 28 days after treatment, the mitochondria appear to have recovered.
Proliferating cell nuclear antigen in normal and regenerating rat livers
1999, Experimental and Molecular Pathology
Immunohistochemical analysis using proliferating cell nuclear antigen (PCNA) antibody shows a negligible number of cells stained in normal liver, but much higher numbers in regenerating liver 24 and 48 h after surgery. We also verified different results by biochemical analysis. Two forms of PCNA, L type (eluted at low concentrations of KCl from a phosphocellulose column) and H type (eluted at high KCl concentrations), were observed in the nucleoplasm of regenerating livers 24 and 48 h after surgery. Treatment of the H type fraction with nuclease caused the H type to disappear and the amount of L type to increase. PCNAs in the cytoplasm are P type (eluted in the pass through fraction) and L type. Surprisingly, the total amounts of P type and L type in cytoplasmic extracts are comparable to those of L type and H type in the nucleoplasm. These results suggest that newly synthesized PCNA is immediately converted into the P and L complex forms. The P type and some of the L type that lacks a nuclear localization signal remain in the cytoplasm; the rest of the L type with a nuclear localization signal is transferred into the nuclei. Then, some of the L type in the nucleoplasm forms the H type, which binds to DNA. These three types of PCNA are also found in significant amounts in the nucleoplasm and cytoplasm of normal rat liver despite its nonproliferating state.
Age-related changes in proliferating cell nuclear antigen levels
1996, Mechanisms of Ageing and Development
To clarify the effect of aging on rat liver regeneration, we compared proliferating cell nuclear antigen (PCNA) levels in control and regenerating livers from young and aged rats 48 h after partial hepatectomy. The nucleoplasm and cytoplasm from regenerating livers of 2-month and 24 month-old rats were fractionated by phosphocellulose column chromatography, aliquots of fractions were transferred to nitrocellulose filters and the amounts of PCNA in each fraction were measured by an immunostaining method. Two forms of PCNA, L type (eluted at low concentrations of KCl) and H type (eluted at high KCl concentrations) were observed in the nucleoplasm from both control and regenerating young rat liver. On the other hand, the cytoplasm contained P type (eluted in the pass-through fraction), L type and H type PCNA. In control liver from aged rats, three types of PCNA in the cytoplasm and two types in the nucleoplasm were present at decreased levels. In regenerating liver from young rats, the increases in L type in the cytoplasm and H type in the nucleoplasm were remarkable. However, none of the three PCNA types increased significantly during liver regeneration in aged rats. Treatment with DNase resulted in the disappearance of the H type with a concomitant increase in the P and L types. These results suggest that the H type is a complex form consisting of the P and L types of PCNA and DNA. These results suggest that the increase in the L type in the cytoplasm reflects newly synthesized PCNA production for cellular proliferation and that the increase in the H type in the nucleoplasm is a reflection of binding to DNA and the fundamental role of PCNA itself in liver regeneration in young rats. On the other hand, there was little increase in any of the three types in regenerating liver from 24-month-old rats. Thus, PCNA content may be closely related to the decrease in the rate of cellular proliferation in aged animals.
p53 functions in the incorporation step in DNA base excision repair in mouse liver mitochondria
2004, Oncogene

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Regular ArticleStimulation of DNA Polymerase γ Activity by Proliferating Cell Nuclear Antigen

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Regular Article
Stimulation of DNA Polymerase γ Activity by Proliferating Cell Nuclear Antigen