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

Surowce polifenolowe. Zastosowania i perspektywy

PRZEDMOWA: "Polifenole są metabolitami wtórnymi roślin i grzybów. Powszechnie występują w kwiatach, korze, korzeniach, łodygach, liściach i owocach roślin. Ich strukturę chemiczną charakteryzuje obecność dwóch lub więcej grup –OH przyłączonych do pierścieni aromatycznych. Do polifenoli zaliczamy lignany, kurkuminoidy, taniny, stilbenoidy, kwasy fenolowe oraz flawonoidy. Ta ostatnia klasa obejmuje: flawony (np. apigenina), flawanony (np. naringenina), flawonole (np. kwercetyna), flawanole (np. katechiny), izoflawony (np. genisteina), antocyjanidyny (np. malwidyna), chalkony (np. buteina), aurony (np. aureuzydyna) i ksantony (np. α-mangostyna). Do tej pory opisano ponad dziesięć tysięcy związków polifenolowych i wciąż nie poznano ich wszystkich. Właściwości polifenoli to temat intensywnie badany na całym świecie ze względu na możliwe wykorzystanie związków polifenolowych w medycynie, farmakologii, kosmetologii, rolnictwie, dietetyce i przemyśle."(...

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

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

eccDNA generation is enhanced in the presence of DNA-damaging agents.

<p>A) Mouse DNA was incubated with mouse nuclear protein 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 and either in absence or presence of 2.5 mg/ml VP-16 (etoposide) B) Quantitative analysis of the result presented in (A), based on 4 independent experiments. All blots were hybridized to MSD probe. C) Formation of eccDNA upon induction of DSB does not require energy and is independent of DNA synthesis. Mouse DNA was incubated with HeLa cytosolic protein extract in the presence of 25 mM EGTA, 2.5 mg/ml VP-16 (etoposide) in the presence or absence of 500 µM ddCTP or 50 µg/ml γ-S-ATP as noted.</p

EccDNA formation <i>in vitro</i>.

<p>A) A scheme of two-dimensional gel electrophoresis (2D gel) showing the migration of linear double strand DNA and relaxed circles. B) Formation of eccDNA from mouse genomic DNA <i>in vitro</i> by mouse nuclear protein extract in the presence of 7 mM MgCl₂, energy-regenerating system (40 mM phosphocreatinine, 10 µg creatine phosphokinase), 4 mM ATP, 250 µM NTPs and 0.5 mM dNTPs (top- hybridization to MSD; bottom- EtBr staining). Note that in addition to eccDNA formation seen upon hybridization, the extract activity caused DNA degradation and thus changed the pattern of linear DNA arc, as is seen from EtBr staining. C). EccDNA is generated in the <i>in vitro</i> reaction and is not liberated from HMW input DNA. Mouse DNA was either digested by <i>EcoRI</i> or subjected to <i>in vitro</i> reaction for eccDNA formation. The blot was hybridized to MSD D). EccDNA produced <i>in vitro</i> consist of major satellite DNA. Products of <i>in vitro</i> reaction (performed similarly to B) were digested either with <i>MnlI</i>, which has 3 recognition sites in MSD or with <i>AluI</i>, which does not cut MSD. The blot was hybridized to total mouse genomic DNA.</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

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