MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage

Abstract

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

Extrachromosomal circular DNA in cancer: history, current knowledge, and methods

“…They often involve damage to the chromosomal DNA and erroneous actions by different DNA repair pathways. For example, two double-strand breaks (DSBs) in the same chromosome can result in a stretch of DNA deleted, which could become circularized (Figure 1A), or secondary DNA loop structures formed in several processes, for example, mismatch repair (MMR) (see Glossary), could be excised and circularized [35][36][37]. Therefore, the mechanisms for eccDNA formation can be different depending on how and where chromatin is subjected to damage and which DNA repair mechanisms are active in a given cell.…”

“…shown to result in a decreased amount of eccDNA in chicken lymphoma and human cancer cells [35,37]. In the most recent of these, deficiency of genes involved in damaged DNA resection and microhomology-mediated end joining (MMEJ) also resulted in decreased eccDNA amounts [37].…”

“…In the most recent of these, deficiency of genes involved in damaged DNA resection and microhomology-mediated end joining (MMEJ) also resulted in decreased eccDNA amounts [37]. In cells lacking proteins essential for NHEJ, both decreased and increased amounts of eccDNA have been reported [22,37,47]. These results might be explained by the different methods used for quantification or a context-or cell-type-dependent role of NHEJ.…”

Extrachromosomal Circular DNA (eccDNA): From Chaos to Function

“…The above studies indicated that the DNA damage repairing pathways including homologous recombination (HR) or nonhomologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) are involved in the formation of microDNAs ( Dillon et al, 2015 ; Paulsen et al, 2021 ). The cells lack 53BP1 and XRCC4 promoting the canonical-NHEJ (c-NHEJ), resulting in more microDNA.…”

“…The cells lack 53BP1 and XRCC4 promoting the canonical-NHEJ (c-NHEJ), resulting in more microDNA. On the contrary, the deficiency of NBS1, POLQ, RAD54, MLH1, MSH2, MSH3, FAN1, and NBS results in the decrease of microDNA ( Paulsen et al, 2021 ). The short, reverted repeats at both ends of junction sites provide perfect places to initialize homologous recombination (HR) during eccDNA formation.…”