We have identified tens of thousands of short extrachromosomal circular DNAs (microDNA) in mouse tissues as well as mouse and human cell lines. These microDNAs are 200–400 bp long, derived from unique non-repetitive sequence and are enriched in the 5' untranslated regions of genes, exons and CpG islands. Chromosomal loci that are enriched sources of microDNA in adult brain are somatically mosaic for micro-deletions that appear to arise from the excision of microDNAs. Germline microdeletions identified by the "Thousand Genomes" project may also arise from the excision of microDNAs in the germline lineage. We have thus identified a new DNA entity in mammalian cells and provide evidence that their generation leaves behind deletions in different genomic loci. Single nucleotide polymorphisms and copy number variations are known sources of genetic variation between individuals (1–5), but there is also great interest in variations that arise during generation of somatic tissues like the mammalian brain, leading to genetic mosaicism between somatic cells. To identify sites of intramolecular homologous recombination during brain development, we searched for extrachromosomal circular DNA (eccDNA) derived from excised chromosomal regions in normal mouse embryonic brains.

SUMMARY MicroDNAs are <400-base extrachromosomal circles found in mammalian cells. Tens of thousands of microDNAs have been found in all tissue types, including sperm. MicroDNAs arise preferentially from areas with high gene density, GC content, and exon density, from promoters with activating chromatin modifications and in sperm from the 5'-UTR of full-length LINE-1 elements, but are depleted from lamin-associated heterochromatin. Analysis of microDNAs from a set of human cancer cell lines revealed lineage-specific patterns of microDNA origins. A survey of microDNAs from chicken cells defective in various DNA repair proteins reveal that homologous recombination and nonhomologous end joining repair pathways are not required for microDNA production. Deletion of the MSH3 DNA mismatch repair protein results in a significant decrease in microDNA abundance, specifically from non-CpG genomic regions. Thus, microDNAs arise as part of normal cellular physiology; either from DNA breaks associated with RNA metabolism or from replication slippage followed by mismatch repair.

Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals.

The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ö zgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and fourway junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2 ⌬B ) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junctionspecific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.Telomeres are nucleoprotein structures that protect the ends of chromosomes and are essential for regulating the replicative lifespan of somatic cells. The DNA component of the mammalian telomere consists of long double-stranded (ds) 2 tracts of the hexameric repeat unit TTAGGG (2) that ends with a G-rich 3Ј single-stranded (ss) overhang (3). Telomeric DNA is thought to be organized into a t-loop "end-capping" structure by the telomere-binding proteins TRF1, TRF2, and POT1 and the proteins that bind to them, TIN2, TPP1, and Rap1 (4, 5). This higher order structure may enable cells to distinguish chromosome ends from random double-strand breaks. Large blocks of telomere repeat sequences can be lost when these end-capping proteins are disrupted, or problems are encountered during DNA replication or repair (for review, see Ref. 6). This typically results in p53-and Rb-mediated senescence or cellular crisis, as evidenced by end-to-end fusions of chromosomes, ATM-dependent activation of p53, and apoptosis (for review, see Ref. 7).Much has been learned about the properties of TRF1 and TRF2 including their binding to DNA and the effects of their ablation or overexpression in the cell. We observed that TRF1 forms filamentous structures on long tracts of telomeric DNA in vitro (8), whereas TRF2 binds preferentially to the telomeric DNA at the junction between the duplex repeats and the ss overhang (9). Both TRF1 and TRF2 contain a similar Myb domain at their COOH terminus that mediates their binding to ds telomeric DNA (10). TRF1 and TRF2 differ in their NH 2 termini, however, which are rich in either acidic residues in TRF1 or basic residues in TRF2. The function of the basic domain of TRF2 is poorly understood. Deletion of this domain (TRF2 ⌬B ) does not impede the DNA binding activity of TRF2 or its localization to telomeres in vivo, but expression of TRF2 ⌬B resulted in stochastic deletions of telomeric DNA, generation of t-loop-sized telomeric circles, cell cycle arrest, and induction of senescence in human cells (11,12). In addition, recent evidence suggested that the...

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mtDNA replication intermediates (mtRIs) are essentially duplex throughout their length, but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mtDNA replication involving RNA incorporation throughout the lagging strand (RITOLS).