Initiation Of DNA Synthesis Research Articles

Structural Basis for Inhibition of Human Primase by Arabinofuranosyl Nucleoside Analogues Fludarabine and Vidarabine.

Nucleoside analogues are widely used in clinical practice as chemotherapy drugs. Arabinose nucleoside derivatives such as fludarabine are effective in the treatment of patients with acute and chronic leukemias and non-Hodgkin’s lymphomas. Although nucleoside analogues are generally known to function by inhibiting DNA synthesis in rapidly proliferating cells, the identity of their in vivo targets and mechanism of action are often not known in molecular detail. Here we provide a structural basis for arabinose nucleotide-mediated inhibition of human primase, the DNA-dependent RNA polymerase responsible for initiation of DNA synthesis in DNA replication. Our data suggest ways in which the chemical structure of fludarabine could be modified to improve its specificity and affinity toward primase, possibly leading to less toxic and more effective therapeutic agents.

Crystal structures of phage NrS-1 N300-dNTPs-Mg2+ complex provide molecular mechanisms for substrate specificity

A novel DNA polymerase from the deep-sea vent phage NrS-1, was characterized as a primase-polymerase (referred to as prim-pol), which works as a self-priming DNA polymerase to synthesize de novo long DNA strands. Functional research on the NrS-1 prim-pol illustrated that the N-terminal 300 residues (referred to as N300) have de novo synthesis activity similar to that of the full-length enzyme. Just like other prim-pols, NrS-1 prim-pol was able to initiate DNA synthesis, proficiently discriminating against ribonucleotides (NTPs), exclusively using deoxynucleotides (dNTPs). However, the structural basis for this discrimination is not well understood. Here, the three kinds of crystal structures of N300-dNTPs-Mg2+ complex were determined. These complex structures shared the identical steric architecture and hydrogen-bond interactions in the catalytic center. The results of biochemical studies indicated that R145 possibly plays an indispensable role in the primer extension. Mutagenesis and structural simulation showed that the backbone carboxyl group of Y146, as a potential sugar selector, was involved in steric clashing with the incoming 2′-OH group of NTPs. However, the mechanism of substrate discrimination probably was different from that of other prim-pols, according to the structural analyses and sequence comparison.

Acinetobacter and Moraxella OriCs are Functional as Chromosomal Replication Origin Transplants in E. coli

Bacterial chromosome replication is triggered by the initiator protein, DnaA, which assembles into a multimeric complex (orisome) that unwinds the replication origin, oriC, and helps to load the replicative DNA helicase. DnaA is highly conserved and the oriCs from nearly all bacteria carry clusters of oriC‐encoded DnaA recognition sites that guide step‐wise and properly timed orisome assembly. However, the arrangement of these sites is widely divergent among bacterial types and prior studies show that heterologous oriCs are only functional in extremely close relatives with nearly identical origin nucleotide sequence. To investigate the basis for DnaA recognition site diversity and possibly identify shared steps in orisome assembly, we replaced E. coli's chromosomal oriC with heterologous donor oriCs from its distant Gammaproteobacterial relatives and measured replication activity in vivo. Despite carrying little sequence similarity to the host, oriCs from Acinetobacter baylyi (ADP1) and Moraxella catarrhalis (ATCC 25240) were active in E. coli and used alternative pathways to assemble E. coli DnaA into functional orisomes. Mapping studies revealed the transplanted oriCs lacked the distinctive DNA unwinding element comprising 13mer repeat motifs as well as the arrayed low affinity DnaA recognition sites that are both hallmarks of E. coli oriC. Using flow cytometry analysis, we found that ADP1 and M. catarrhalis oriCs were also unable to initiate DNA synthesis at the proper cell cycle time in their E. coli hosts. Thus, the mechanisms for initiation timing must be distinct from those that permit replication fork assembly on these origins. We propose that E. coli and ADP1‐type orisomes share a sub‐architecture required for mechanical function (unwinding and helicase loading), but also carry type‐specific variations in the arrangement and affinity of DnaA recognition sites that are required for proper initiation timing using their cognate DnaAs. We propose that replication origin diversity reflects the many varieties of cell cycle‐coupled regulatory mechanisms used to control the availability/accessibility of DnaA at the oriCs of different bacterial types. Further studies of DnaA‐oriC interactions on heterologous origins may also reveal novel targets for broad spectrum bacterial growth inhibitors as well as those for type‐specific inhibition.Support or Funding InformationNational Institutes of Health (GM54042 to AL) and Florida Institute of Technology Holzer Lequear Fund for Molecular Genetics (to JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Random Priming: Labeling of Purified DNA Fragments by Extension of Random Oligonucleotides.

In this protocol, oligonucleotides that are heterogeneous in sequence serve as primers for the initiation of DNA synthesis on single-stranded templates. Labeled dNTPs ([α-32P]dNTPs or biotin-, DIG-, or fluorescein-labeled dUTPs) are incorporated into the new DNA by the Klenow fragment of Escherichia coli DNA polymerase I (Pol I). The method can be adapted to radiolabel DNA in slices cut from gels cast with low-melting-temperature agarose.

RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Temperature-sensitive origin-binding protein as a tool for investigations of herpes simplex virus activities in vivo.

While it is fairly clear that herpes simplex virus (HSV) DNA replication requires at least seven virus-encoded proteins in concert with various host cell factors, the mode of this process in infected cells is still poorly understood. Using HSV-1 mutants bearing temperature-sensitive (ts) lesions in the UL9 gene, we previously found that the origin-binding protein (OBP), a product of the UL9 gene, is only needed in the first 6 hours post-infection. As this finding was just a simple support for the hypothesis of a biphasic replication mode, we became convinced through these earlier studies that the mutants tsR and tsS might represent suitable tools for more accurate investigations in vivo. However, prior to engaging in highly sophisticated research projects, knowledge of the biochemical features of the mutated versions of OBP appeared to be essential. The results of our present study demonstrate that (i) tsR is most appropriate for cell biological studies, where only immediate early and early HSV gene products are being expressed without the concomital viral DNA replication, and (ii) tsS is a prime candidate for the analysis of HSV DNA replication processes because of its reversibly thermosensitive OBP-ATPase, which allows one to switch on the initiation of DNA synthesis precisely.

An archaeal primase functions as a nanoscale caliper to define primer length

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper.

Design, synthesis and biological evaluation of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)benzoyl hydrazide derivatives as thymidylate synthase (TS) inhibitors and as potential antitumor drugs

The Inhibition of cellular nucleotide metabolism to promote apoptosis is a key principle of cancer therapy. Thymidylate synthase (TS) is a key rate-limiting enzyme in the initiation of DNA synthesis in cell. Here, we presented two types of thymidylate synthase inhibitors, and, the key pharmacological properties of these two types of thymidylate synthase inhibitor were extracted and combined to design new compounds with inhibitory activity. Therefore, two series of 42 new compounds with the common biological effect of promoting apoptosis are designed and synthesized by combination principle. Most of the compounds had good anti-proliferative activity on A549, OVCAR-3, SGC7901 and MDA-MB-231 cells. The IC50 of compound 10l on A549 cells was 1.26 μM, which was better than that of pemetrexed (PTX, IC50 = 3.31 μM), furthermore, the selection index of compound 10l was higher than PTX. Flow cytometry analysis showed that compound 10l (the apoptosis rate is 39.4%) could induce A549 cell apoptosis and effectively inhibit tumor cell proliferation. Further western blot analysis showed that compound 10l could induce intrinsic apoptosis by activating caspase-3, increasing expression of cleaved caspase-3 and reducing the ratio of bcl-2/bax. All of this makes compound 10l to be a promising compound in future animal tumor models.

P-270 - Phenomenology and outcomes of acute antioxidative stress in normal human cells

A number of clinical trials have shown harmful effects of high-dosage antioxidant (AO) interventions. Antioxidative stress defined as a negative impact of pharmacological AOs on cells and tissues is one of the concepts being discussed in this regard. However, the number of experimental studies on this topic is quite limited. As a rule, experimentally revealed negative impact of high AO doses on cells is associated with a variety of side-effects of each individual substance. Thereby, the basic question that arises when trying to generalize published results is as follows: is it possible to determine the acute antioxidative stress as a specific type of damaging effect arising from the abrupt lowering of the basal ROS level in cells? If yes, what are the basic features and outcomes of this stress? Here we propose our point of view, basing on the analysis of cell response to high doses of AOs of different origin and type of activity. We show that all tested AOs can block cell proliferation, hamper both initiation and progression of DNA synthesis, lead to DNA strand breaks accumulation, and disturb regulation of DNA synthesis in normal human stem and non-stem cell cultures. The outcomes of the stress depend on the cell type.

Role of Cyclin A2 In Aging Hippocampus And rRna Dynamics

BackgroundNovel functions of cyclin A2 (CCNA2) have been demonstrated including DNA synthesis initiation and progression, DNA repair, and protein translation through RNA binding. Previously, we reported that loss of CCNA2 leads to hippocampal defects characterized by abnormal DNA repair and impaired performance on behavioral tests due to memory and learning deficits. Abnormalities in RNA processing and synaptic plasticity are mechanisms associated with deficits in memory and aging. This study was addressed to investigate effects of cyclin A2 loss on modulation of RNA processing in the hippocampus of aged mice.MethodsAll work involving animals was performed under the auspices of a protocol approved by the Ohio State University Institutional Animal Care and Use Committee. CamkIIα‐cre mice were interbred with CCNA2fl/fl mice to achieve adult ablation of CCNA2 in the hippocampus and cerebral cortex. To evaluate brain morphology, animals were sacrificed at 8 months. After fixed in 4% paraformaldehyde, crysections were obtained at 12 μm. Electron microscopy was performed and number of synapses and polyribosomes were quantified.ResultsUltrastructural analysis of Stratum Moleculare (SM) and Reticulare (SR) in 8mo mices showed a decrease of the synapse density in CamkIIαcre CCNA2 fl/fl mices as compared with control. However, quantity of ribosomes was increased only in SM of the hippocampus in mutant mices. Immunostaining for ribosomal RNA (rRNA) revealed an accumulation of rRNA granules in dentate gyrus and Cornu Ammonis (CA) both regions of the hippocampus. Quantitative analysis resulted an increased rRNA in the SR. To elucidate if mechanisms of translational regulation are modified by the lack of CCNA2 we screened for alterations of 40S ribosomal protein S6 (S6), and translational repressor eukaryotic initiation factor 4E binding protein (ELF4E) and cytoplasmic polyadenylation element binding protein 1 (CPEB1).ConclusionsOur data shows a distinct role for CCNA2 in adult hippocampus in the regulation of synaptic density as well as the prevention of rRNA granule formation. Alterations in RNA dynamics caused by loss of CCNA2 may be implicated in neuronal disorders and neurodegeneration.Support or Funding InformationThis work was sponsored by NIH/NHLBI grants RO1 HL132354This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Furanone derivatives as new inhibitors of CDC7 kinase: development of structure activity relationship model using 3D QSAR, molecular docking, and in silico ADMET

Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays a vital role in the replication initiation of DNA synthesis. Overexpression of the CDC7 in various tumor growths and in cell proliferation makes it a promising target for treatment of cancers. To investigate the binding between the CDC7 and furanone inhibitors, and in order to design highly potent inhibitors, a three-dimensional quantitative structure activity relationship (3D-QSAR) with molecular docking was performed. The optimum CoMSIA model showed significant statistical quality on all validation methods with a determination coefficient (R2 = 0.945), bootstrapping R2 mean (BS-R2 = 0.960), and leave-one-out cross-validation (Q2) coefficient of 0.545. The predictability of this model was evaluated by external validation using a test set of nine compounds with a predicted determination coefficient R2test of 0.96, besides the mean absolute error (MAE) of the test set was 0.258 log units. The extracted contour maps were used to identify the important regions, where the modification was necessary to design a new molecule with improved activity. Furthermore, a good consistency between the molecular docking and contour maps strongly demonstrates that the molecular modeling is reliable. Based on those obtained results, we designed several new potent CDC7 inhibitors, and their inhibitory activities were validated by the molecular models. Additionally, those newly designed inhibitors showed promising results in the preliminary in silico ADMET evaluations.

SJP-L-5 inhibits HIV-1 polypurine tract primed plus-strand DNA elongation, indicating viral DNA synthesis initiation at multiple sites under drug pressure

In a previous study the small molecule SJP-L-5 that inhibits HIV replication, has been shown to block uncoating of the viral capsid. Continued study showed that SJP-L-5 might hinder HIV capsid uncoating by blocking the completion of reverse transcription. However, to date, the mechanism has not been fully elucidated. Here, the effects of SJP-L-5 for reverse transcription were explored via quantitative PCR, DIG-labelled ELISA, fluorescent resonance energy transfer, and Southern blot assays. We also analyzed the resistance profile of this compound against reverse transcriptase. Our results show that SJP-L-5 preferentially inhibits PPT primed plus-strand DNA synthesis (EC50 = 13.4 ± 3.0 μM) over RNA primed minus-strand DNA synthesis (EC50 > 3,646 μM), resulting in formation of five segmented plus-strand DNA and loss of HIV DNA flap, suggesting failure of both nuclear import and integration. Moreover, resistance study evidenced that SJP-L-5 requires the amino acid residues Val108 and Tyr181 to exert an inhibitory effect. These results indicate SJP-L-5 as a new non-nucleoside reverse transcriptase inhibitor that inhibits HIV-1 polypurine tract primed plus-strand DNA synthesis, initiating HIV-1 down-stream plus-strand DNA synthesis at multiple sites under drug pressure.

High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6pmol/106cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350pmol/106cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

A Proximity Ligation-Based Method for Quantitative Measurement of D-Loop Extension in S. cerevisiae.

Homologous recombination faithfully restores the sequence information interrupted by a DNA double-strand break by referencing an intact DNA molecule as a template for repair DNA synthesis. DNA synthesis is primed from 3'-OH end of the invading DNA strand in the displacement loop (D-loop). Here, we describe a simple and quantitative proximity ligation-based assay to study the initiation of homologous recombination-associated DNA synthesis initiated at the D-loop and final product formation. The D-loop extension assay overcomes the semiquantitative nature and some limitations of the current PCR-based technique and facilitates the study of the recombination-associated DNA synthesis.

The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box.

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress.

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18-/- and Polκ-/- cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18-Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.

Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence E. coli genomic DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends.

DNA replication and mismatch repair safeguard against metabolic imbalances.

During DNA replication, the major DNA replicative polymerases Polδ and Pole introduce noncomplementary nucleotides approximately once in every 100,000 polymerization events. Proofreading properties of these polymerases improve their fidelity by 10- to 100-fold, and postreplicative DNA mismatch repair (MMR), which acts as a spell checker to remove mismatches that escape polymerase proofreading, improves this fidelity even further, resulting in mutation rates as low as 2 × 10−10 substitutions per base per cell division (1). Mutations in genes encoding MMR proteins and DNA polymerase proofreading activities are linked to elevated mutation rates and diseases such as cancer, and these rates are synergistically increased in double mutants (2, 3). In PNAS, Schmidt et al. (4) describe work in which they screened for defects in genome stability in genetic backgrounds where processes that limit mutagenesis were compromised. Their work provides new insights for how cellular metabolism and nucleotide pool homeostasis interact to avoid mutation and maintain genome stability. They also provide evidence that DNA polymerase fidelity and MMR functions are sufficiently robust to compensate at least partially for defects in cellular metabolism (Fig. 1). Fig. 1. Removing the buffering capabilities of DNA replication and DNA MMR uncovers new factors that contribute to nucleotide pool homeostasis. Multiple mechanisms contribute to the overall fidelity of DNA replication. For example, each of the four mechanisms outlined in this figure can compensate for defects in the others. ( i ) A homeostasis mechanism responds to cellular metabolism to regulate the biosynthesis of dNTP and dNTP precursors to maintain a balanced concentration of dNTPs. ( ii ) DNA polymerase proofreading activities: During eukaryotic DNA replication, the major replicative polymerases Pole and Polδ both contain proofreading activities that enable them to excise misincorporation errors. However, Polα, which initiates DNA synthesis at origins and at Okazaki fragments, does not contain such a function. … [↵][1]1To whom correspondence should be addressed. Email: eea3{at}cornell.edu. [1]: #xref-corresp-1-1

In Vivo DNA Re-replication Elicits Lethal Tissue Dysplasias

Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated invivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication.

Genomic rearrangements induced by unscheduled DNA double strand breaks in somatic mammalian cells.

DNA double-strand breaks (DSBs) are highly toxic lesions that can lead to profound genome rearrangements and/or cell death. They routinely occur in genomes due to endogenous or exogenous stresses. Efficient repair systems, canonical non-homologous end-joining and homologous recombination exist in the cell and not only ensure the maintenance of genome integrity but also, via specific programmed DNA double-strand breaks, permit its diversity and plasticity. However, these repair systems need to be tightly controlled because they can also generate genomic rearrangements. Thus, when DSB repair is not properly regulated, genome integrity is no longer guaranteed. In this review, we will focus on non-programmed genome rearrangements generated by DSB repair, in somatic cells. We first discuss genome rearrangements induced by homologous recombination and end-joining. We then discuss recently described rearrangement mechanisms, driven by microhomologies, that do not involve the joining of DNA ends but rather initiate DNA synthesis (microhomology-mediated break-induced replication, fork stalling and template switching and microhomology-mediated template switching). Finally, we discuss chromothripsis, which is the shattering of a localized region of the genome followed by erratic rejoining.