Endo-reduplication in mouse liver after conditional mutation of ORC2 and combined mutation of ORC1 and ORC2.

The six subunit Origin Recognition Complex (ORC) is essential for loading MCM2-7 at origins of DNA replication to promote initiation of DNA replication in organisms ranging from S. cerevisiae to humans. In rare instances, as in cancer cell-lines in culture with mutations in ORC1 , ORC2 or ORC5 , or in endo-reduplicating mouse hepatocytes in vivo without ORC1 , DNA replication has been observed in the virtual absence of individual ORC subunits. Although ORC1 is dispensable in the mouse liver for endo-reduplication, because of the homology of ORC1 with CDC6, it could be argued that CDC6 was substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2 , to demonstrate that mouse embryo fibroblasts require ORC2 for proliferation, but that the mouse hepatocytes can carry out DNA synthesis in vitro and endo-reduplicate in vivo , despite the deletion of ORC2 . Combining the conditional mutation of ORC1 and ORC2 revealed that the mouse liver can still carry out endo-reduplication despite the deletion of the two genes, both during normal development and after partial hepatectomy. Since endo-reduplication, like normal S phase replication, requires the presence of MCM2-7 on the chromatin, these results suggest that in primary hepatocytes there is a mechanism to load sufficient MCM2-7 to carry out effective DNA replication despite the virtual absence of two subunits of ORC.

Integrative analysis of DNA replication origins and ORC-/MCM-binding sites in human cells reveals a lack of overlap.

Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae. Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.

sRNA-Effector: A tool to expedite discovery of small RNA regulators.

microRNAs (miRNAs) are small regulatory RNAs that repress target mRNA transcripts through base pairing. Although the mechanisms of miRNA production and function are clearly established, new insights into miRNA regulation or miRNA-mediated gene silencing are still emerging. In order to facilitate the discovery of miRNA regulators or effectors, we have developed sRNA-Effector, a machine learning algorithm trained on enhanced crosslinking and immunoprecipitation sequencing and RNA sequencing data following knockdown of specific genes. sRNA-Effector can accurately identify known miRNA biogenesis and effector proteins and identifies 9 putative regulators of miRNA function, including serine/threonine kinase STK33, splicing factor SFPQ, and proto-oncogene BMI1. We validated the role of STK33, SFPQ, and BMI1 in miRNA regulation, showing that sRNA-Effector is useful for identifying new players in small RNA biology. sRNA-Effector will be a web tool available for all researchers to identify potential miRNA regulators in any cell line of interest.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

Modeling and monitoring the effects of three partly conserved Ile residues in the dimerization domain of a Mip-like virulence factor from Escherichia coli.

FKBP22, an Escherichia coli-made peptidyl-prolyl cis-trans isomerase, has shown considerable homology with Mip-like virulence factors. While the C-terminal domain of this enzyme is used for executing catalytic function and binding inhibitor, the N-terminal domain is employed for its dimerization. To precisely determine the underlying factors of FKBP22 dimerization, its structural model, developed using a suitable template, was carefully inspected. The data show that the dimeric FKBP22, like dimeric Mip proteins, has a V-like shape. Further, it dimerizes using 40 amino acid residues including Ile 9, Ile 17, Ile 42, and Ile 65. All of the above Ile residues except Ile 9 are partly conserved in the Mip-like proteins. To confirm the roles of the partly conserved Ile residues, three FKBP22 mutants, constructed by substituting them with an Ala residue, were studied as well. The results together indicate that Ile 65 has little role in maintaining the dimeric state or enzymatic activity of FKBP22. Conversely, both Ile 17 and Ile 42 are essential for preserving the structure, enzymatic activity, and dimerization ability of FKBP22. Ile 42 in particular looks more essential to FKBP22. However, none of these two Ile residues is required for binding the cognate inhibitor. Additional computational studies also indicated the change of V-shape and the dimeric state of FKBP22 due to the Ala substitution at position 42. The ways Ile 17 and Ile 42 protect the structure, function, and dimerization of FKBP22 have been discussed at length.Communicated by Ramaswamy H. Sarma.

Integrative analysis of DNA replication origins and ORC/MCM binding sites in human cells reveals a lack of overlap.

Based on experimentally determined average inter-origin distances of ∼100 kb, DNA replication initiates from ∼50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the Origin Recognition Complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and 5 ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ∼7.5 million union origins identified by all datasets, only 0.27% were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques (20,250 shared origins), suggesting extensive variability in origin usage and identification. 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF binding sites, G-quadruplex sites and activating histone marks, these overlaps are comparable or less than that of known Transcription Start Sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ∼13,000 reproducible ORC binding sites in human cancer cells, and only 4.5% were within 1 kb of the ∼11,000 union MCM2-7 binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, S. cerevisiae . Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.

XRN2 suppresses aberrant entry of tRNA trailers into argonaute in humans and Arabidopsis.

MicroRNAs (miRNAs) are a well-characterized class of small RNAs (sRNAs) that regulate gene expression post-transcriptionally. miRNAs function within a complex milieu of other sRNAs of similar size and abundance, with the best characterized being tRNA fragments or tRFs. The mechanism by which the RNA-induced silencing complex (RISC) selects for specific sRNAs over others is not entirely understood in human cells. Several highly expressed tRNA trailers (tRF-1s) are strikingly similar to microRNAs in length but are generally excluded from the microRNA effector pathway. This exclusion provides a paradigm for identifying mechanisms of RISC selectivity. Here, we show that 5' to 3' exoribonuclease XRN2 contributes to human RISC selectivity. Although highly abundant, tRF-1s are highly unstable and degraded by XRN2 which blocks tRF-1 accumulation in RISC. We also find that XRN mediated degradation of tRF-1s and subsequent exclusion from RISC is conserved in plants. Our findings reveal a conserved mechanism that prevents aberrant entry of a class of highly produced sRNAs into Ago2.

Cryptic lncRNA-encoded ORFs: A hidden source of regulatory proteins.

A majority of the human genome is transcribed into noncoding RNAs, of which long noncoding RNAs (lncRNAs) form a large and heterogeneous fraction. While lncRNAs are mostly noncoding, recent evidence suggests that cryptic translation within some lncRNAs may produce proteins with important regulatory functions. In this issue of the JCI, Zheng, Wei, and colleagues used an integrative functional genomic strategy to systematically identify cryptic lncRNA-encoded ORFs that play a role in estrogen receptor-positive (ER+) breast cancer (BC). They identified 758 cryptic lncRNA-encoded ORFs undergoing active translation, of which 28 had potential functional and clinical relevance in ER+ BC. The LINC00992-encoded polypeptide GT3-INCP was upregulated in ER+ BC and drove tumor growth. GT3-INCP was regulated by estrogen and the ER and acted via the transcription factor GATA3 to regulate BC susceptibility and risk genes. These findings discern a largely unexplored class of molecules and have implications for many pathologies, including cancer.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1.

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (-821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

Sequence driven interaction of amino acids in de-novo designed peptides determines c-Myc G-quadruplex unfolding inducing apoptosis in cancer cells.

c-MYC proto-oncogene harbors a putative G-quadruplex structure (Pu27) at the NHEIII1 domain, which can shuffle between transcriptional inhibitor quadruplex and transcriptionally active duplex. In cancer cells this quadruplex destabilization is preferred and NHEIII1 domain assume a duplex topology thereby inducing c-MYC overexpression and tumorigenesis. Hence, the c-MYC quadruplex acts as an excellent target for anti-cancer therapy. Though researcher have tried to develop G-quadruplex targeted small molecules, work with G-quadruplex targeting peptides is very limited. Here we present a peptide that can bind to c-MYC quadruplex, destabilize the tetrad core, and permit the formation of a substantially different structure from the quartet core seen in the canonical G-quadruplexes. Such conformation potentially acted as a roadblock for transcription factors thereby reducing cMYC expression. This event sensitizes the cancer cell to activate apoptotic cascade via the c-MYC-VEGF-A-BCL2 axis. This study provides a detailed insight into the peptide-quadruplex interface that encourages better pharmacophore design to target dynamic quadruplex structure. We believe that our results will contribute to the development, characterization, and optimization of G-quadruplex binding peptides for potential clinical application.

Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer.

Background: Bladder cancer (BLCA) is a common and deadly disease that results in a reduced quality of life for the patients and a significant economic burden on society. A better understanding of tumorigenesis is needed to improve clinical outcomes. Recent evidence places the RNA modification m1A and its regulatory proteins TRMT6/TRMT61A and ALKBH3 in BLCA pathogenesis. Methods: TRMT6/TRMT61A, ALKBH1, and ALKBH3 expression was examined in human BLCA cell lines and a normal urinary tract epithelium cell line through qRT-PCR and western blot analysis. Prestoblue Cell Viability Reagent, wound-healing assay, and live-cell imaging-based cell displacement analysis, were conducted to assess proliferation, migration, and displacement of this BLCA cell line panel. Cell survival was assessed after inducing cellular stress and activating the unfolded protein response (UPR) with tunicamycin. Moreover, siRNA-mediated gene silencing in two BLCA cell lines (5637 and HT1197) was conducted to investigate the biological roles of TRMT6/TRMT61A. Results: Heterogeneous morphology, proliferation, displacement, tunicamycin sensitivity, and expression levels of m1A regulators were observed among the panel of cell lines examined. In general, TRMT61A expression was increased in BLCA cell lines when compared to SV-HUC-1. Depletion of TRMT6/TRMT61A reduced proliferation capacity in both 5637 and HT1197 cell lines. The average cell displacement of 5637 was also reduced upon TRMT6/TRMT61A depletion. Interestingly, TRMT6/TRMT61A depletion decreased mRNA expression of targets associated with the ATF6-branch of the UPR in 5637 but not in HT1197. Moreover, cell survival after induction of cellular stress was compromised after TRMT6/TRMT61A knockdown in 5637 but not in HT1197 cells. Conclusion: The findings suggest that TRMT6/TRMT61A plays an oncogenic role in BLCA and is involved in desensitizing BLCA cells against cellular stress. Further investigation into the regulation of TRMT6/TRMT61A expression and its impact on cellular stress tolerance may provide insights for future BLCA treatment.

Characterization of novel small non-coding RNAs and their modifications in bladder cancer using an updated small RNA-seq workflow.

Background: Bladder cancer (BLCA) is one of the most common cancer types worldwide. The disease is responsible for about 200,000 deaths annually, thus improved diagnostics and therapy is needed. A large body of evidence reveal that small RNAs of less than 40 nucleotides may act as tumor suppressors, oncogenes, and disease biomarkers, with a major focus on microRNAs. However, the role of other families of small RNAs is not yet deciphered. Recent results suggest that small RNAs and their modification status, play a role in BLCA development and are promising biomarkers due to their high abundance in the exomes and body fluids (including urine). Moreover, free modified nucleosides have been detected at elevated levels from the urine of BLCA patients. A genome-wide view of small RNAs, and their modifications, will help pinpoint the molecules that could be used as biomarker or has important biology in BLCA development. Methods: BLCA tumor tissue specimens were obtained from 12 patients undergoing transurethral resection of non-muscle invasive papillary urothelial carcinomas. Genome-wide profiling of small RNAs less than 40 bases long was performed by a modified protocol with TGIRT (thermostable group II reverse transcriptase) to identify novel small RNAs and their modification status. Results: Comprehensive analysis identified not only microRNAs. Intriguingly, 57 ± 15% (mean ± S.D.) of sequencing reads mapped to non-microRNA-small RNAs including tRNA-derived fragments (tRFs), ribosomal RNA-derived fragments (rRFs) and YRNA-derived fragments (YRFs). Misincorporation (mismatch) sites identified potential base modification positions on the small RNAs, especially on tRFs, corresponding to m1A (N1-methyladenosine), m1G (N1-methylguanosine) and m2 2G (N2, N2-dimethylguanosine). We also detected mismatch sites on rRFs corresponding to known modifications on 28 and 18S rRNA. Conclusion: We found abundant non-microRNA-small RNAs in BLCA tumor samples. Small RNAs, especially tRFs and rRFs, contain modifications that can be captured as mismatch by TGIRT sequencing. Both the modifications and the non-microRNA-small RNAs should be explored as a biomarker for BLCA detection or follow-up.

tRForest: a novel random forest-based algorithm for tRNA-derived fragment target prediction.

tRNA fragments (tRFs) are small RNAs comparable to the size and function of miRNAs. tRFs are generally Dicer independent, are found associated with Ago, and can repress expression of genes post-transcriptionally. Given that this expands the repertoire of small RNAs capable of post-transcriptional gene expression, it is important to predict tRF targets with confidence. Some attempts have been made to predict tRF targets, but are limited in the scope of tRF classes used in prediction or limited in feature selection. We hypothesized that established miRNA target prediction features applied to tRFs through a random forest machine learning algorithm will immensely improve tRF target prediction. Using this approach, we show significant improvements in tRF target prediction for all classes of tRFs and validate our predictions in two independent cell lines. Finally, Gene Ontology analysis suggests that among the tRFs conserved between mice and humans, the predicted targets are enriched significantly in neuronal function, and we show this specifically for tRF-3009a. These improvements to tRF target prediction further our understanding of tRF function broadly across species and provide avenues for testing novel roles for tRFs in biology. We have created a publicly available website for the targets of tRFs predicted by tRForest.

Function and Therapeutic Implications of tRNA Derived Small RNAs.

tRNA derived small RNAs are mainly composed of tRNA fragments (tRFs) and tRNA halves (tiRs). Several functions have been attributed to tRFs and tiRs since their initial characterizations, spanning all aspects of regulation of the Central Dogma: from nascent RNA silencing, to post-transcriptional gene silencing, and finally, to translational regulation. The length distribution, sequence diversity, and multifaceted functions of tRFs and tiRs positions them as attractive new models for small RNA therapeutics. In this review, we will discuss the principles of tRF biogenesis and function in order to highlight their therapeutic potential.

Integrated bioinformatic pipeline using whole-exome and RNAseq data to identify germline variants correlated with cancer.

Germline Variants (GVs) are effective in predicting cancer risk and may be relevant in predicting patient outcomes. Here we provide a bioinformatic pipeline to identify GVs from the TCGA lower grade glioma cohort in Genomics Data Commons. We integrate paired whole exome sequences from normal and tumor samples and RNA sequences from tumor samples to determine a patient's GV status. We then identify the subset of GVs that are predictive of patient outcomes by Cox regression. For complete details on the use and execution of this protocol, please refer to Chatrath et al. (2019) and Chatrath et al. (2020).

TRMT6/61A-dependent base methylation of tRNA-derived fragments regulates gene-silencing activity and the unfolded protein response in bladder cancer.

RNA modifications are important regulatory elements of RNA functions. However, most genome-wide mapping of RNA modifications has focused on messenger RNAs and transfer RNAs, but such datasets have been lacking for small RNAs. Here we mapped N1-methyladenosine (m1A) in the cellular small RNA space. Benchmarked with synthetic m1A RNAs, our workflow identified specific groups of m1A-containing small RNAs, which are otherwise disproportionally under-represented. In particular, 22-nucleotides long 3' tRNA-fragments are highly enriched for TRMT6/61A-dependent m1A located within the seed region. TRMT6/61A-dependent m1A negatively affects gene silencing by tRF-3s. In urothelial carcinoma of the bladder, where TRMT6/61A is over-expressed, higher m1A modification on tRFs is detected, correlated with a dysregulation of tRF targetome. Lastly, TRMT6/61A regulates tRF-3 targets involved in unfolded protein response. Together, our results reveal a mechanism of regulating gene expression via base modification of small RNA.

Lipid Clustering in Mycobacterial Cell Envelope Layers Governs Spatially Resolved Solvation Dynamics.

The mycobacterial cell envelope acts as a multilayered barrier to drugs. However, the role of lipid composition in the properties of different mycobacterial membranes, otherwise dictating their interactions with drugs, is poorly understood. In this study, we found that hydration states, solvation relaxation kinetics, rotational lipid mobility, and lateral lipid diffusion differed between inner and outer mycobacterial membranes. Molecular modeling showed that lipid clustering patterns governed membrane dynamics in the different layers of the cell envelope. By regulating membrane properties, lipid composition and structure modulated water abundance and interactions with lipid head groups. These findings can help deepen our understanding of the physical chemistry underlying membrane structure and function, as well as the interaction of mycobacterial membranes with drugs and host membranes.

Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors.

Many lncRNAs have been discovered using transcriptomic data; however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as DRReRNA) acts as an enhancer RNA for the Myod1 gene in cis and stimulates the expression of other promyogenic genes in trans by recruiting the cohesin complex. Here, experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by prediction algorithms in the absence of experimental information. We show that these specific and structurally distinct domains are required for induction of promyogenic genes, for binding genomic sites and gene expression regulation, and for binding the cohesin complex. Myod1 induction and cohesin interaction comprise only a subset of MUNC phenotype. Our study reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs.

The tumor-suppressive long noncoding RNA DRAIC inhibits protein translation and induces autophagy by activating AMPK.

Long noncoding RNAs (lncRNAs) are long RNA transcripts that do not code for proteins and have been shown to play a major role in cellular processes through diverse mechanisms. DRAIC, a lncRNA that is downregulated in castration-resistant advanced prostate cancer, inhibits the NF-κB pathway by inhibiting the IκBα kinase. Decreased DRAIC expression predicted poor patient outcome in gliomas and seven other cancers. We now report that DRAIC suppresses invasion, migration, colony formation and xenograft growth of glioblastoma-derived cell lines. DRAIC activates AMP-activated protein kinase (AMPK) by downregulating the NF-κB target gene GLUT1, and thus represses mTOR, leading to downstream effects, such as a decrease in protein translation and increase in autophagy. DRAIC, therefore, has an effect on multiple signal transduction pathways that are important for oncogenesis, namely, the NF-κB pathway and AMPK-mTOR-S6K/ULK1 pathway. The regulation of NF-κB, protein translation and autophagy by the same lncRNA explains the tumor-suppressive role of DRAIC in different cancers and reinforces the importance of lncRNAs as emerging regulators of signal transduction pathways. This article has an associated First Person interview with the first author of the paper.

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

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.