Replication Independent Formation of Extrachromosomal Circular DNA in Mammalian Cell-Free System

Extrachromosomal circular DNA (eccDNA) is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence- independent enzymes as human protein extract can produce mouse- specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg2+ and is enhanced by double strand DNA breaks

Role of PP2Cα in cell growth, in radio- and chemosensitivity, and in tumorigenicity

<p>Abstract</p> <p>Background</p> <p>PP2Cα is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFβ-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Cα in cell growth and in radio- and chemosensitivity, wild type and PP2Cα siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Cα in tumorigenesis.</p> <p>Results</p> <p>It was found that knockdown of PP2Cα did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Cα siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Cα siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased.</p> <p>Conclusion</p> <p>Based on these findings, we conclude that PP2Cα is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Cα may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer.</p

Protein Phosphatase Magnesium Dependent 1A (PPM1A) Plays a Role in the Differentiation and Survival Processes of Nerve Cells

The serine/threonine phosphatase type 2C (PPM1A) has a broad range of substrates, and its role in regulating stress response is well established. We have investigated the involvement of PPM1A in the survival and differentiation processes of PC6-3 cells, a subclone of the PC12 cell line. This cell line can differentiate into neuron like cells upon exposure to nerve growth factor (NGF). Overexpression of PPM1A in naive PC6-3 cells caused cell cycle arrest at the G2/M phase followed by apoptosis. Interestingly, PPM1A overexpression did not affect fully differentiated cells. Using PPM1A overexpressing cells and PPM1A knockdown cells, we show that this phosphatase affects NGF signaling in PC6-3 cells and is engaged in neurite outgrowth. In addition, the ablation of PPM1A interferes with NGF-induced growth arrest during differentiation of PC6-3 cells

EccDNA – generating enzymes are not sequence-specific.

<p>Human genomic DNA was incubated with either heat-inactivated or native mouse nuclear protein extract. The samples were separated on 2D gel, blotted and hybridized to total human DNA probe. Mouse genomic DNA was incubated with either mouse (inactivated or native) or human nuclear protein extract, under the conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006126#pone-0006126-g001" target="_blank">Fig.1B</a>. The samples were separated on 2D gel, blotted and hybridized to MSD probe.</p

Formation of eccDNA <i>in vitro</i> from an artificial substrate.

<p>TAR vector containing ∼35 kb MSD insert was incubated with HeLa cytosolic extract under conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006126#s2" target="_blank">Materials and Methods</a> in the presence of 25 mM EGTA without additional supplies. The blot was hybridized to MSD.</p

Residual level of magnesium is sufficient for eccDNA formation.

<p>A) eccDNA formation depends on trace amounts of ions present in protein/DNA preparations. Mouse DNA was incubated with mouse nuclear protein extract under conditions described in 1B either in the presence or absence of Mg²⁺ and 25 mM EDTA. Top- hybridization, bottom- EtBr staining. B) Chelation with EGTA does not affect the reaction. The reactions were performed similarly to (A) in the absence or presence of 25 mM EGTA. Top- hybridization, bottom- EtBr staining. All blots were hybridized to MSD probe.</p

The reaction does not require energy supplement.

<p>A) Formation of eccDNA in the absence of energy input. Mouse DNA was incubated with mouse nuclear protein extract under the conditions described in 1B, either in the presence or absence of energy regenerating system and ATP. B) Effect of γ-S-ATP on the reaction. The reactions were performed similarly to (A) in the absence or presence of 50 µg/ml γ-S-ATP and EGTA. The blots were hybridized to MSD probe.</p

Formation of eccDNA <i>in vitro</i> by cytosolic extract.

<p>Mouse genomic DNA was incubated with Hela cytosolic extract under conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006126#s2" target="_blank">Materials and Methods</a> and the presence of 25 mM EGTA without additional supplies. The blot was hybridized to MSD.</p