EccDNA formation is dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA double-strand break

Teressa Paulsen, Pumoli Malapati, Rebeka Eki, Tarek Abbas, Anindya Dutta

Preprint posted on 4 December 2020

DNA double-strand breaks induce extrachromosomal circular DNA formation.

Selected by Sree Rama Chaitanya


Our cells have genetic material other than linear chromosomes (like mitochondrial DNA). But recent pieces of evidence reveal the existence of free circular DNA called extrachromosomal circular DNA (eccDNA or ecDNA, also called double minutes) in normal and cancer cells. These can impel genetic variability in isogenic cells or tumors, causing intra-tumor heterogeneity to perpetuate drug-resistant colonies. ecDNA could also harbor protein-coding oncogenes or even non-canonical small RNA genes. ecDNA form when DNA breaks produce linear DNA molecules either by excision or by copying, which circularize by ligation. However, the molecular mechanisms and pathways that lead to the formation of ecDNA are not clear. Therefore, the authors of the current preprint set forward to investigate the possible DNA repair pathways that contribute to ecDNA biogenesis.


General strategy (fig.1). The authors evaluated previously identified ecDNA molecules in human (U2OS, 293) and chicken (DT40) cells using inverse quantitative PCR (check supplementary tables of the preprint for the list of ecDNAs used in this study). They purify the circles from cell lysates using commercial circular plasmid prep kits followed by extensive digestion with exonucleases to degrade any contaminating linear DNA. They designed outward-facing primers that will amplify across the junction sequence of a circle but will not produce an amplicon from linear DNA. They used mitochondrial DNA as a control for normalization. In this way, they deem to achieve ecDNA specific amplicons.

1. Assay developed by the authors to quantify ecDNA.

Key findings

  1. First, they found that 293 cells manifested a two-fold increase in ecDNAs when exposed to different exogenous sources of DNA damage (like UV, cisplatin, neocarzinostatin, etc).
  2. Then, they tested if one DNA double-strand break (DSB) is sufficient to induce ecDNA formation locally (fig.2). For this purpose, they induced a DSB using CRISPR at a genomic locus (Chr22:18624104). This locus usually has low levels of ecDNA and contains unique sequences. Its transcriptional output is also low to avoid any confounding effects. The guide-RNA was site-specific, and the cutting efficiency was greater than 60%. Using a set of outward-directed primers, they were able to report ecDNA molecules that span about 150-1500bp DNA adjoining the DSB-site. They were not able to detect ecDNA, at DNAs greater than1500bp from the DSB-site, suggesting ecDNA form at closer proximity to the DSB. Thus, they suggest that a single DSB is sufficient to induce ecDNA molecules in cis.
    2. Diagram of induced DSBs at locus Chr22:18624104 in 293A cells and the amplification of ecDNA arising proximal to the cut site using outward-facing primers. Quantification of ecDNA isolated 48 hours after the transfection of a p413 vector containing CAS9 and a gRNA sequence or a gRNA sequence targeting Chr22:18624104 or Chr12:117100086.


  3. To dissect the DNA repair pathways involved in ecDNA biology, they used isogenic cell lines lacking DNA repair genes (or chemical inhibitors of DNA repair enzymes) (fig.3). They broadly classified the genes as the ones that can repair broken DNA through end-resection or not. In these lines, they demonstrate that depletion of canonical non-homologous end joining (c-NHEJ) genes induced ecDNA molecules in different cell lines. Additionally, cells depleted of gene products that are part of the end-resection pathway (like NBS1) manifested lower ecDNA molecules. Moreover, cells lacking proteins of the microhomology-mediated end joining (MMEJ) and mismatch repair (MMR) pathways manifested lower levels of ecDNA. While proteins of base excision repair pathway (BER) (like FEN1, APE1, etc.) do not seem to impact ecDNA levels, inhibition of poly (ADP-ribose) polymerases (PARPs), a common component of MMEJ and BER pathways reduced ecDNA levels. Additionally, chemical inhibition of RAD52 (single-strand annealing, SSA) and ERCC1-XPF (nucleotide excision repair, NER) did not impact ecDNA levels significantly.

    3. DNA repair genes knocked-out or inhibited in this work. Graphical summary of whether mutants or inhibitors or a given repair pathway increased (green) or decreased (red) ecDNA levels.
  4. Interestingly, the authors found a mixed impact on ecDNA levels when proteins of the homologous recombination pathway (HR) were down-regulated. Here, except for RAD54 and BRCA1, other HR proteins (RAD51, BRCA2, and CTIP) did not show a significant reduction in ecDNA levels. Notably, when they induced DSBs chemically (using neocarzinostatin), c-NHEJ deficient cells manifested higher levels of ecDNA, and MMEJ deficient cells had lower levels of ecDNAs. Altogether, the authors suggest that while MMEJ (and resection) supports, c-NHEJ could thwart ecDNA formation (fig.3)

    4. End-resection-independent pathway of repair is active in G1, while end-resection (and homology) dependent repair is more active in S and G2.
  5. Repair of damaged DNA is very much dependent on cell cycle stages. While c-NHEJ can occur in all phases of the cell cycle, alternative-NHEJ pathways that favor end-resection (like MMEJ, HR) preferentially occur during the replicating S phase (fig.4). Hence, the authors chemically arrested the cells (using hydroxyurea and nocodazole) and evaluated ecDNA levels in different cell cycle stages. They show that ecDNA tends to accumulate in cells during S-, G2-, and M-phases. Also, inhibition of replication (using aphidicolin) reduced ecDNA levels, reinforcing that ecDNA tend to form during S/G2 phases. But intriguingly, ecDNA levels dropped when cells re-enter the G1-phase.


Genome instability is a hallmark of cancer. However, the aftermath of drugs targeted to DNA repair pathways is often catastrophic on healthy cells, and severely impacts an individual’s recovery. The equation is complex, and solving the cancer puzzle is not just simply summing the parts. Hence, it is important to understand how genome instability specifically drives tumor progression and triggers intra-tumor heterogeneity.

5. Proposed model of endogenous ecDNA formation from a single DSB.

Here, the authors propose that even a single DSB is sufficient to trigger ecDNA in replicating cells (fig.5). ecDNA could be a way out for cancer cells to make extra copies of oncogenes or drug-resistance genes. This warrants a better understanding of the outcomes of genome instability so that we can develop smarter therapeutic combinations to promote faster and relapse-free recovery.

 Acknowledgments: I am thankful to all the authors for their support, and Anindya Dutta for taking the time to comment on the preLight and replying promptly.

All figures and figure legends of this preLight are taken and modified directly from Paulsen et. al., 2020 under a CC-BY 4.0 international license.



Tags: dna damage, extrachromosomal circular dna, genome instability

Posted on: 5 January 2021


Read preprint (No Ratings Yet)

Author's response

Anindya Dutta (AD) shared

1. It is very interesting that ecDNAs are lost when cells exit mitosis and enter the G1-phase (Fig.4). What do the authors speculate about this data? Are ecDNA molecules digested or re-integrated into the chromosomes?

AD: We think that they are lost in the cytoplasm or digested in the cytoplasm.

2. How do the authors think their model fits or explains ecDNA formation (if any) in postmitotic cells like muscle and neuron cells?

AD: The formation of circles from single double-strand breaks, suggests that such breaks (which occur from superoxides) in postmitotic cells may give rise to ecDNA.

3. The authors’ smartly used CRISPR to induce a specific DSB and evaluate ecDNA in cis. Would the authors consider using enzyme-based systems to induce DSBs at a specific gene locus6 and measure ecDNA?

AD: Yes, other methods of generating site-specific breaks should be tested, especially methods that produce staggered DNA ends.

4. The authors picked a non-transcribing site for CRISPR-induced DSB to avoid any confounding effect. Did the authors measure any break-induced transcription activity after the CRISPR, especially considering your earlier work that transcriptional activity could be important for ecDNA formation7?

AD: We have not yet done so.

5. Do the authors think if primers used here can be used for quantitative imaging to observe if the specific cells that lack DNA repair genes (like MMEJ, MMR) produce more ecDNA?

AD: Imaging the ecDNA as they are being produced will indeed be very useful.

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cancer biology category:

Also in the cell biology category:

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage