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

Teressa Paulsen,Pumoli Malapati,Tarek Abbas,Yoshiyuki Shibata,Mouadh Benamar,Briana Wilson,Anindya Dutta,Rebeka Eki

doi:10.1093/nar/gkab984

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.

Highlights

The field of extrachromosomal circular DNA has recently burgeoned due to the increasing evidence of both the prevalence and active role of eccDNA in normal tissues and cancers [1–8]
To determine the changes in eccDNA abundance under different conditions, we developed a quantitative assay that detects the levels of specific candidate sequences that represent the most abundant microDNA molecules detected by nextgeneration sequencing of microDNA in our previous rolling circle amplification (RCA) libraries (Figure 1A, Supplemental Table S1; NCBI GEO, GSE68644 and GSE36088)
The results show that the microDNA obtained from different mutant cell lines and after different chemical treatments generally have characteristics common among themselves and consistent with previously published microDNA, e.g. GC content, presence of microhomology at the ligation junction (Supplemental Figure S1F–H)

Summary

Introduction

The field of extrachromosomal circular DNA (eccDNA) has recently burgeoned due to the increasing evidence of both the prevalence and active role of eccDNA in normal tissues and cancers [1–8]. The larger eccDNA (>10 kb), termed double minutes, have been well categorized and studied due to their contribution to cancer growth by sustaining and amplifying full gene sequences through circular molecules that contain promoters, enhancers and replication origin sequences They are often composed of DNA segments from multiple loci (multilocular) and their massive size enables these molecules to be visible through fluorescent microscopy during normal karyotyping [9–11]. The eccDNAs of smaller sizes (99% of the eccDNA population in normal and cancer cells [5,12–15] These eccDNA molecules, termed microDNA, are highly heterogeneous, are usually derived from a single locus (monolocular) and have been found to express microRNA and si-like-RNA which are capable of affecting gene expression [5]. We focus on the microDNA, excluding the very large eccDNA, double minutes

Methods

Results

Conclusion