eccDNA: Insights Into Formation, Distribution, and Disease

Extrachromosomal circular DNA (eccDNA) is genetic material found outside the standard chromosomal framework in eukaryotic cells. Once considered rare, eccDNA is now recognized as widespread and functionally significant, influencing genome stability, gene expression, and disease development.

Understanding its formation, distribution, and potential role in aging and disease has become a key area of research.

Mechanisms Of Formation

The generation of eccDNA arises from various genomic processes, including DNA repair, replication errors, and chromosomal instability. A primary pathway involves the erroneous repair of double-strand breaks (DSBs). When DNA damage occurs, repair mechanisms such as non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) can excise chromosomal fragments, which then circularize. Studies show that repair-associated eccDNAs often contain repetitive sequences, indicating that regions with high repeat density are particularly prone to circularization.

Replication stress also contributes to eccDNA formation. Stalled replication forks can generate DNA fragments that fail to reintegrate into the genome and instead form circles. Oncogenes and regulatory elements frequently appear in eccDNA, suggesting that replication stress may preferentially generate functionally relevant sequences. This has been observed in cancer cells, where eccDNA amplifies oncogenic signals, promoting tumor heterogeneity and drug resistance.

Transposable elements further influence eccDNA biogenesis. Mobile genetic elements such as LINEs and SINEs can drive genomic rearrangements that lead to excision and circularization. Increased transposase activity correlates with higher eccDNA formation, particularly under genomic stress. Homologous recombination between repetitive sequences can also facilitate eccDNA excision, emphasizing the role of repetitive elements in its dynamics.

Structural Features

EccDNA varies in size, sequence composition, and topology, affecting its biological functions. These DNA circles range from small, sub-kilobase fragments to large megabase-sized structures. Smaller eccDNAs, often called microDNAs, arise mainly from non-coding regions and tend to be enriched in CpG islands and promoter sequences, suggesting a role in gene regulation. Larger eccDNAs, including double minutes (DMs) in cancer, frequently contain amplified oncogenes, altering gene dosage and driving tumor progression.

The circular topology of eccDNA makes it resistant to exonucleolytic degradation, allowing it to persist in cells, particularly in dividing populations where it can be asymmetrically inherited. EccDNA can adopt supercoiled, relaxed, or multimeric conformations, with supercoiling enhancing its interaction with transcriptional machinery. Studies using nanopore sequencing and electron microscopy reveal that eccDNAs often contain palindromic sequences and repetitive elements, which may facilitate circularization and influence transcriptional activity.

The epigenetic landscape of eccDNA also plays a role in its function. Unlike chromosomal DNA, eccDNA exhibits a unique chromatin structure that enhances accessibility to transcription factors. Chromatin immunoprecipitation sequencing (ChIP-seq) studies show that eccDNA is often enriched in active histone marks, such as H3K27ac and H3K4me3, indicating a transcriptionally permissive state. This open chromatin configuration allows eccDNA to serve as an additional transcriptional template, which can lead to oncogene amplification and contribute to intratumoral heterogeneity and therapeutic resistance.

Tissue Distribution

EccDNA prevalence and composition vary by tissue type, influenced by cellular turnover rates, metabolic activity, and genomic architecture. Rapidly dividing tissues, such as bone marrow and intestinal epithelium, exhibit higher eccDNA levels due to frequent DNA replication and repair events. In contrast, post-mitotic tissues like the brain have lower eccDNA abundance, though neuron-specific eccDNAs have been identified, suggesting roles in neural plasticity and gene expression.

The liver, a metabolically active organ exposed to oxidative stress, harbors eccDNA populations reflecting its dynamic genomic environment. Oxidative damage can induce DNA breaks, promoting eccDNA formation as a repair byproduct. Skeletal muscle, despite being largely non-proliferative, contains eccDNA enriched in repetitive sequences, raising questions about its persistence and role in cellular homeostasis.

In reproductive tissues, eccDNA exhibits distinct characteristics that may influence germline stability. Sperm cells carry eccDNA fragments, suggesting potential intergenerational transmission. In female reproductive tissues, eccDNA has been detected in endometrial and ovarian cells, with some studies linking its accumulation to hormone-driven genomic changes. The tissue-specific variability of eccDNA underscores its diverse biological roles, from transient genomic artifacts to stable regulatory elements.

Age-Related Changes

EccDNA accumulation increases with age, reflecting declining DNA repair efficiency and accumulated replication stress. This trend appears in both mitotic and post-mitotic tissues, suggesting that eccDNA formation results from more than just cell division. The persistence of eccDNA in aging cells raises questions about its role in cellular senescence, a state of permanent growth arrest with altered gene expression.

The sequence composition of eccDNA also changes with age, with an enrichment of repetitive elements and transposable sequences. This shift likely results from increased genomic instability, as repetitive regions are more prone to DNA breaks and rearrangements. Age-related epigenetic modifications may further influence which genomic regions are excised to form eccDNA, potentially disrupting transcriptional activity and contributing to age-associated phenotypes such as reduced regenerative capacity and altered metabolism.

Detection Techniques

Identifying and characterizing eccDNA requires specialized methodologies due to its structural differences from linear genomic DNA. Traditional sequencing techniques often overlook eccDNA, necessitating targeted approaches that preserve its circular nature while minimizing contamination from chromosomal fragments.

A widely used method involves exonuclease treatments, which selectively degrade linear DNA while preserving circular DNA. This enhances the specificity of eccDNA isolation, allowing for more accurate assessments of its abundance and composition.

Rolling circle amplification (RCA) exploits eccDNA’s circular topology to generate high-molecular-weight concatemers for sequencing. High-throughput sequencing approaches, such as Circle-Seq and ATAC-seq adaptations, provide genome-wide insights into eccDNA distribution and epigenetic modifications. Long-read platforms like nanopore and PacBio sequencing enable the resolution of complex eccDNA structures, revealing amplification and rearrangement patterns. These techniques have been instrumental in uncovering the functional significance of eccDNA in normal and diseased states.

Associations With Disease

EccDNA has been linked to various diseases, with cancer being the most extensively studied. Tumor cells frequently harbor high eccDNA levels, often containing amplified oncogenes such as MYC and EGFR. Unlike chromosomal amplifications, which are constrained by linear genomic architecture, eccDNA enables dynamic gene dosage alterations, contributing to tumor heterogeneity and adaptive resistance to therapy. Single-cell sequencing studies reveal that eccDNA can be asymmetrically distributed during cell division, leading to subpopulations of cancer cells with distinct genetic advantages. This phenomenon has been observed in glioblastomas and neuroblastomas, where eccDNA-driven oncogene amplification correlates with aggressive disease progression and poor clinical outcomes.

Beyond cancer, eccDNA has been implicated in neurodegenerative disorders and cardiovascular diseases. In Alzheimer’s disease, eccDNA derived from repetitive elements has been detected in affected brain regions, suggesting a role in genomic instability and neuronal dysfunction. Similarly, eccDNA accumulation in atherosclerotic plaques has been proposed as a biomarker for vascular aging and disease progression. The ability of eccDNA to modulate gene expression and interact with cellular pathways highlights its potential as both a diagnostic marker and a therapeutic target. Ongoing research aims to clarify its contributions to disease pathology and explore interventions that mitigate its effects.