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

Abstract

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.

Molecular characterization of cell-free eccDNAs in human plasma

“…So far, knowledge on mechanisms of eccDNA generation is limited. Proposed repair mechanisms include both the replication-based mechanisms and non-replicative mechanisms 36 – 40 . The non-replicative repair mechanisms mainly include NAHR, NHEJ and MMEJ.…”

Extrachromosomal Circular DNA: Current Knowledge and Implications for CNS Aging and Neurodegeneration

“…As aforementioned, it is suggested that genomic instability, apart from DNA replication and replication-related repair attempts, are primarily involved. Accordingly, augmented eccDNA load is found as a result of double-strand break (DSB) induction [36], which thus might operate as a trigger for eccDNA formation both in a physiological and pathological context.…”

“…Of note, several lines of evidence indicate that eccDNA biogenesis does not absolutely depend on homologous recombination (HR) but can be produced by excision from chromosomal sequences, e.g., during the process of HR-independent DNA repair [36]. It has even been suggested that neither HR nor non-homologous end joining (NHEJ; or classical NHEJ, c-NHEJ) are required for eccDNA production and that the eccDNA amount is weakly correlated with the proliferation rate of an individual tissue type [9].…”

“…This notion gains support from recent characterizations of the repertoire of DNA-repair mechanisms engaged in eccDNA formation. As a basic proof of principle connecting eccDNA with an HR-restricted neural milieu, Cohen and Lavi demonstrated that eccDNA release from high molecular weight DNA can principally be independent of de novo DNA synthesis and HR but will alternatively rely on chromatin excision [36]. This finding is emphasized by the requirement of active DNA ligase IV, a major enzyme necessary to ligate DSB in NHEJ, in the formation of eccDNA in mammalian cells [37].…”

Cell-Cycle-Associated Amplified Genomic-DNA Fragments (CAGFs) Might Be Involved in Chloroquine Action and Resistance in Plasmodium falciparum

“…CAGFs belong to extrachromosomal ssDNA molecules and their copy numbers are increased after CQ treatment [11], which distinguishes them from double-stranded extrachromosomal circular DNA (eccDNA) molecules found in all eukaryotes [39] [40]. Although eccDNA molecules can be generated by sublethal drug exposure [41] and levels of total eccDNA molecules have been elevated after exposure of organism to genotoxic agent [42], the copy number of every single eccDNA molecule can not be increased after exposure to genotoxic agent because the formation of eccDNA molecules is through excision of chromosomal sequences and no de-novo DNA synthesis is involved [43]. The eccDNA-related excision of chromosomal sequences could be considered as a kind of genoautotomy in which nonlethal double-stranded genomic-DNA fragments are excised from chromosomes [14].…”