FGF2-induced upregulation of DNA polymerase-δ p12 subunit in endothelial cells

Dell’Era, Patrizia; Nicoli, Stefania; Peri, Giuseppe; Nieddu, Mariella; Ennas, Maria Grazia; Presta, Marco

doi:10.1038/sj.onc.1208359

Cited by 12 publications

(8 citation statements)

References 31 publications

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“…Our findings suggest that PDIP38 may possess functions in the mitochondria that may not be related to its ability to associate with pol ␦ and PCNA. In this regard, P12, the fourth subunit of pol ␦, was recently suggested to possess additional, cell proliferation-unrelated functions (61). Our results raise the possibility that PDIP38 may have functions that include interaction with the nuclear DNA replication system or in the linkage between pol ␦ and viral DNA replication.…”

Section: Discussionmentioning

confidence: 64%

Further Characterization of Human DNA Polymerase δ Interacting Protein 38

Xie

Wang

et al. 2005

Journal of Biological Chemistry

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Polymerase ␦ interacting protein 38 (PDIP38) was identified as a human DNA polymerase (pol) ␦ interacting protein through a direct interaction with p50, the small subunit of human pol ␦. 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 ␦ activity in vitro and interacted with human papillomavirus 16 E7 oncoprotein, suggesting that PDIP38 might play a role in the pol ␦-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 ␦-mediated DNA replication or DNA repair under certain conditions such as viral infection.Chromosomal DNA replication is a tightly regulated process, which requires multiple protein complexes working in a highly coordinated manner. In eukaryotic cells, three major DNA polymerases, DNA polymerases ␣, ␦, and ⑀, are involved in DNA replication (1-3). Although it is clear that the DNA polymerase ␣/primase is involved in initiating both leading and lagging strand synthesis, the precise roles of DNA polymerase (pol) 1 ␦ and DNA polymerase ⑀ remain to be fully defined. A mutant without the catalytic domain of the large subunit of pol ⑀ in Schizosaccharomyces pombe was found to be viable (4), and pol ␦ and pol ␣ were sufficient to replicate both the leading and lagging strands of double-stranded DNA in an SV40 DNA replication system (5). These earlier studies pointed to the fact that pol ␦ was likely to be the major polymerase for the elongation of both leading and lagging strands of chromosomal DNA in eukaryotic cells. This was supported by recent studies that showed that there was inefficient elongation of both leading and lagging DNA strands in Xenopus egg extracts depleted of pol ␦ (6). pol ⑀ was suggested to play a role mainly in leading strand DNA synthesis (6). pol ␦, working together with Fen1 and proliferating cell nuclear antigen (PCNA), is required for Okazaki fragment maturation, and the 3Ј-5Ј-exonuclease activity of pol ␦ is important for this process (7-9). pol ␦ is also involved in telomerase-mediated telomere addition in vivo (10) and participates in several DNA repair pathways (11, 12). The ability of pol ␦ to ...

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Section: Discussionmentioning

confidence: 64%

Further Characterization of Human DNA Polymerase δ Interacting Protein 38

Xie

Wang

et al. 2005

Journal of Biological Chemistry

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“…Although it is tempting to consider that p12 depletion may be involved in the inhibition of DNA synthesis that is observed after DNA damage with UV (see below), this may be too simplistic an explanation. It has been reported that p12 silencing in mouse endothelial cells causes a significant decrease in FGF2 (fibroblast growth factor)-stimulated cell growth (48). In these cells, p12 expression is specifically up-regulated by FGF2 but not by other mitogenic stimuli (48).…”

Section: Discussionmentioning

confidence: 99%

“…It has been reported that p12 silencing in mouse endothelial cells causes a significant decrease in FGF2 (fibroblast growth factor)-stimulated cell growth (48). In these cells, p12 expression is specifically up-regulated by FGF2 but not by other mitogenic stimuli (48). Although the findings that p12 levels can affect cell proliferation are consistent with the hypothesis that this is due to effects on DNA synthesis via the modification of Pol ␦, a defect in Pol ␦ The ability of Pol ␦ 3-p12 to perform processive DNA synthesis was examined by its ability to extend a single primer on M13 DNA.…”

Section: Discussionmentioning

confidence: 99%

A Novel DNA Damage Response

Zhang

Zhou

Trusa

et al. 2007

Journal of Biological Chemistry

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Mammalian DNA polymerase (Pol) ␦ is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol ␦ in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol ␦ by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol ␦ function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR ؊/؊ cells but not in ATM ؊/؊ cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol ␦ in both DNA replication and DNA repair.Three eukaryotic DNA polymerases, ␣, ␦, and ⑀, are involved in chromosomal DNA replication (1, 2). Pol 2 ␦ (3) and Pol ⑀ possess proofreading 3Ј to 5Ј exonuclease activities, which allows them to replicate DNA with high fidelity (1, 2). Mammalian Pol ␦ consists of a tightly associated dimer of the 125-kDa catalytic subunit and p50 (4, 5) that is associated with the p68 (6 -8) and p12 (9) subunits. The current model for DNA synthesis at the replication fork is that Pol ␣/primase synthesizes RNA primers plus short stretches of DNA. These are then elongated to Okazaki fragments of ϳ200 nucleotides by Pol ␦ on the lagging strand of the replication fork (1, 2). Leading strand synthesis requires highly processive synthesis and was originally thought to require Pol ␦, since the latter was able to perform the replication of the SV40 genome in an in vitro system (10). The role of Pol ⑀ has been extensively studied in yeast, where it is essential for viability in both Saccharomyces cerevisiae and Schizosaccharomyces pombe (1, 2). However, it is the C-terminal checkpoint domain and not the N-terminal catalytic domain that is essential (i.e. Pol ⑀ activity per se is dispensable, and it has been suggested that Pol ␦ activity may be able to replace Pol ⑀ activity) (11,12). In mammalian cells, it has been found that although Pol ␦ alone can replicate SV40 DNA replication, both Pol ␦ and Pol ⑀ are involved in cellular DNA replication (13). Localization and cross-linking studies suggest that Pol ␦ and Pol ⑀ may function independently of each other and that their involvement in ch...

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“…A previous study showed that the POLD4 ortholog of Cdm1 in Schizosaccharomyces pombe (S. pombe) is a nonessential gene (20), although it may be required in mammalian cells (21,22). To study human POLD4 functions, we depleted POLD4 using siD4 and analyzed the cell cycle progression.…”

Section: Pold4 Required For Cell Cycle Progressionmentioning

confidence: 99%

Regulation of DNA Polymerase POLD4 Influences Genomic Instability in Lung Cancer

et al. 2010

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Genomic instability is an important factor in cancer susceptibility, but a mechanistic understanding of how it arises remains unclear. We examined hypothesized contributions of the replicative DNA polymerase δ (pol δ) subunit POLD4 to the generation of genomic instability in lung cancer. In examinations of 158 lung cancers and 5 mixtures of 10 normal lungs, cell cycle-and checkpoint-related genes generally showed mRNA expression increases in cancer, whereas POLD4 showed reduced mRNA in small cell lung cancer (SCLC). A fraction of non-small cell lung cancer patients also showed low expression comparable with that in SCLC, which was associated with poor prognosis. The lung cancer cell line ACC-LC-48 was found to have low POLD4 expression, with higher histone H3K9 methylation and lower acetylation in the POLD4 promoter, as compared with the A549 cell line with high POLD4 expression. In the absence of POLD4, pol δ exhibited impaired in vitro DNA synthesis activity. Augmenting POLD4 expression in cells where it was attenuated altered the sensitivity to the chemical carcinogen 4-nitroquinoline-1-oxide. Conversely, siRNA-mediated reduction of POLD4 in cells with abundant expression resulted in a cell cycle delay, checkpoint activation, and an elevated frequency of chromosomal gap/break formation. Overexpression of an engineered POLD4 carrying silent mutations at the siRNA target site rescued these phenotypes, firmly establishing the role of POLD4 in these effects. Furthermore, POLD4 overexpression reduced intrinsically high induction of γ-H2AX, a well-accepted marker of double-stranded DNA breaks. Together, our findings suggest that reduced expression of POLD4 plays a role in genomic instability in lung cancer. Cancer Res; 70(21); 8407-16. ©2010 AACR.

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FGF2-induced upregulation of DNA polymerase-δ p12 subunit in endothelial cells

Cited by 12 publications

References 31 publications

Further Characterization of Human DNA Polymerase δ Interacting Protein 38

Further Characterization of Human DNA Polymerase δ Interacting Protein 38

A Novel DNA Damage Response

Regulation of DNA Polymerase POLD4 Influences Genomic Instability in Lung Cancer

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