Numerous DNA Double-strand Breaks Research Articles

Chromosomal Rearrangements and Chromothripsis: The Alternative End Generation Model

Chromothripsis defines a genetic phenomenon where up to hundreds of clustered chromosomal rearrangements can arise in a single catastrophic event. The phenomenon is associated with cancer and congenital diseases. Most current models on the origin of chromothripsis suggest that prior to chromatin reshuffling numerous DNA double-strand breaks (DSBs) have to exist, i.e., chromosomal shattering precedes rearrangements. However, the preference of a DNA end to rearrange in a proximal accessible region led us to propose chromothripsis as the reaction product of successive chromatin rearrangements. We previously coined this process Alternative End Generation (AEG), where a single DSB with a repair-blocking end initiates a domino effect of rearrangements. Accordingly, chromothripsis is the end product of this domino reaction taking place in a single catastrophic event.

From Double-Strand Break Recognition to Cell-Cycle Checkpoint Activation: High Content and Resolution Image Cytometry Unmasks 53BP1 Multiple Roles in DNA Damage Response and p53 Action.

53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53–53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.

De novo deletions and duplications at recombination hotspots in mouse germlines

Numerous DNA double-strand breaks (DSBs) arise during meiosis to initiate homologous recombination. These DSBs are usually repaired faithfully, but here, we uncover a distinct type of mutational event in which deletions form via joining of ends from two closely spaced DSBs (double cuts) within a single hotspot or at adjacent hotspots on the same or different chromatids. Deletions occur in normal meiosis but are much more frequent when DSB formation is dysregulated in the absence of the ATM kinase. Events between chromosome homologs point to multi-chromatid damage and aborted gap repair. Some deletions contain DNA from other hotspots, indicating that double cutting at distant sites creates substrates for insertional mutagenesis. End joining at double cuts can also yield tandem duplications or extrachromosomal circles. Our findings highlight the importance of DSB regulation and reveal a previously hidden potential for meiotic mutagenesis that is likely to affect human health and genome evolution.

Combination BET Family Protein and HDAC Inhibition Synergistically Elicits Chondrosarcoma Cell Apoptosis Through RAD51-Related DNA Damage Repair.

BackgroundChondrosarcoma is the second-most common type of bone tumor and has inherent resistance to conventional chemotherapy. Present study aimed to explore the therapeutic effect and specific mechanism(s) of combination BET family protein and HDAC inhibition in chondrosarcoma.MethodsTwo chondrosarcoma cells were treated with BET family protein inhibitor (JQ1) and histone deacetylase inhibitors (HDACIs) (vorinostat/SAHA or panobinostat/PANO) separately or in combination; then, the cell viability was determined by Cell Counting Kit-8 (CCK-8) assay, and the combination index (CI) was calculated by the Chou method; cell proliferation was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation and colony formation assay; cell apoptosis and reactive oxygen species (ROS) level were determined by flow cytometry; protein expressions of caspase-3, Bcl-XL, Bcl-2, γ-H2AX, and RAD51 were examined by Immunoblotting; DNA damage was determined by comet assay; RAD51 and γ-H2AX foci were observed by immunofluorescence.ResultsCombined treatment with JQ1 and SAHA or PANO synergistically suppressed the growth and colony formation ability of the chondrosarcoma cells. Combined BET and HDAC inhibition also significantly elevated the ROS level, followed by the activation of cleaved-caspase-3, and the downregulation of Bcl-2 and Bcl-XL. Mechanistically, combination treatment with JQ1 and SAHA caused numerous DNA double-strand breaks (DSBs), as evidenced by the comet assay. The increase in γ-H2AX expression and foci formation also consistently indicated the accumulation of DNA damage upon cotreatment with JQ1 and SAHA. Furthermore, RAD51, a key protein of homologous recombination (HR) DNA repair, was found to be profoundly suppressed. In contrast, ectopic expression of RAD51 partially rescued SW 1353 cell apoptosis by inhibiting the expression of cleaved-caspase-3.ConclusionTaken together, our results disclose that BET and HDAC inhibition synergistically inhibit cell growth and induce cell apoptosis through a mechanism that involves the suppression of RAD51-related HR DNA repair in chondrosarcoma cells.

Kinetochores, cohesin, and DNA breaks: Controlling meiotic recombination within pericentromeres.

In meiosis, DNA break formation and repair are essential for the formation of crossovers between homologous chromosomes. Without crossover formation, faithful meiotic chromosome segregation and sexual reproduction cannot occur. Crossover formation is initiated by the programmed, meiosis‐specific introduction of numerous DNA double‐strand breaks, after which specific repair pathways promote recombination between homologous chromosomes. Despite its crucial nature, meiotic recombination is fraud with danger: When positioned or repaired inappropriately, DNA breaks can have catastrophic consequences on genome stability of the resulting gametes. As such, DNA break formation and repair needs to be carefully controlled. Within centromeres and surrounding regions (i.e., pericentromeres), meiotic crossover recombination is repressed in organisms ranging from yeast to humans, and a failure to do so is implicated in chromosome missegregation and developmental aneuploidy. (Peri)centromere sequence identity and organization diverge considerably across eukaryotes, yet suppression of meiotic DNA break formation and repair appear universal. Here, we discuss emerging work that has used budding and fission yeast systems to study the mechanisms underlying pericentromeric suppression of DNA break formation and repair. We particularly highlight a role for the kinetochore, a universally conserved, centromere‐associated structure essential for chromosome segregation, in suppressing (peri)centromeric DNA break formation and repair. We discuss the current understanding of kinetochore‐associated and chromosomal factors involved in this regulation and suggest future avenues of research.

S1-seq Assay for Mapping Processed DNA Ends.

During meiosis, the specialized cell division giving rise to gametes, numerous DNA double-strand breaks (DSBs) are introduced at multiple places throughout the genome by the topoisomerase-like protein Spo11. Homologous recombination, a highly conserved DSB repair pathway, is employed for their repair and ensures the formation of chiasmata and the proper segregation of homologous chromosomes. In the initial steps of recombination, end resection takes place, wherein Spo11 is endonucleolytically released and the 5'-terminal strands of each DSB are exonucleolytically processed, exposing the ssDNA necessary to identify a homologous repair template. DNA removed by DSB processing is reconstituted by DNA synthesis, which copies genetic information from the intact homologous template. We developed a next-generation sequencing assay, termed S1-seq, to study DNA end resection genome-wide at high-spatial resolution during yeast meiotic recombination. The assay relies on the fact that removal of the ssDNA tails of resected DSBs marks the position where resection stopped. Molecular features of resection are revealed by sequencing of these ssDNA-to-dsDNA junctions and comparison to high-resolution Spo11 DSB maps. We describe the experimental and computational methods for S1-seq as applied to meiosis in the SK1 strain of budding yeast Saccharomyces cerevisiae and discuss how it can also be applied to map DSBs and recombination intermediates.

Genes, Proteins, and Biological Pathways Preventing Chromothripsis.

The highly complex structural genome variations chromothripsis, chromoanasynthesis, and chromoplexy are subsumed under the term chromoanagenesis, which means chromosome rebirth. Precipitated by numerous DNA double-strand breaks, they differ in number of and distances between breakpoints, associated copy number variations, order and orientation of segments, and flanking sequences at joining points. Results from patients with the autosomal dominant cancer susceptibility disorder Li-Fraumeni syndrome implicated somatic TP53 mutations in chromothripsis. TP53 participates in the G2/M phase checkpoint, halting cell cycling after premature chromosome compaction during the second half of the S phase, thus preventing chromosome shattering. By experimental TP53 ablation and micronucleus induction, one or a few isolated chromosomes underwent desynchronized replication and chromothripsis. Secondly, chromothripsis occurred after experimental induction of telomere crisis after which dicentric chromosomes sustained TREX1-mediated resolution of chromosome bridges and kataegis. Third, DNA polymerase Polθ-dependent chromothripsis has been documented. Finally, a family with chromothripsis after L1 element-dependent retrotransposition and Alu/Alu homologous recombination has been reported. Human chromosomal instability syndromes share defects in responses to DNA double-strand breaks, characteristic cell cycle perturbations, elevated rates of micronucleus formation, premature chromosome compaction, and apoptosis. They are also associated with elevated susceptibility to malignant disease, such as medulloblastomas and gliomas in ataxia-telangiectasia, leukemia and lymphoma in Bloom syndrome, and osteosarcoma and soft tissue sarcoma in Werner syndrome. The latter syndrome is characterized by a premature aging-like progressive decline of mesenchymal tissues. In all thus far studied cases, constitutional chromothripsis occurred in the male germline and male patients with defects in the double-strand break response genes ATM, MRE11, BLM, LIG4, WRN, and Ku70 show impaired fertility. Conceivably, chromothripsis may, in a stochastic rather than deterministic way, be implicated in germline structural variation, malignant disease, premature aging, genome mosaicism in somatic tissues, and male infertility.

Tel1(ATM)-mediated interference suppresses clustered meiotic double-strand-break formation.

Meiotic recombination is a critical step in gametogenesis for many organisms, enabling the creation of genetically diverse haploid gametes. In each meiotic cell, recombination is initiated by numerous DNA double-strand breaks (DSBs) created by Spo11, the evolutionarily conserved topoisomerase-like protein, but how these DSBs are distributed relatively uniformly across the four chromatids that make up each chromosome pair is poorly understood. Here we employ Saccharomyces cerevisiae to demonstrate distance-dependent DSB interference in cis (in which the occurrence of a DSB suppresses adjacent DSB formation)--a process that is mediated by the conserved DNA damage response kinase, Tel1(ATM). The inhibitory function of Tel1 acts on a relatively local scale, while over large distances DSBs have a tendency to form independently of one another even in the presence of Tel1. Notably, over very short distances, loss of Tel1 activity causes DSBs to cluster within discrete zones of concerted DSB activity. Our observations support a hierarchical view of recombination initiation where Tel1(ATM) prevents clusters of DSBs, and further suppresses DSBs within the surrounding chromosomal region. Such collective negative regulation will help to ensure that recombination events are dispersed evenly and arranged optimally for genetic exchange and efficient chromosome segregation.

Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity

The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC50 in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.

Targeting RAD51 phosphotyrosine-315 to prevent unfaithful recombination repair in BCR-ABL1 leukemia

AbstractChronic myeloid leukemia chronic phase (CML-CP) CD34+ cells contain numerous DNA double-strand breaks whose unfaithful repair may contribute to chromosomal instability and disease progression to blast phase (CML-BP). These phenomena are often associated with the appearance of imatinib-resistant BCR-ABL1 kinase mutants (eg, T315I) and overexpression of BCR-ABL1. Here we show that BCR-ABL1 (nonmutated and T315I mutant) promoted RAD51 recombinase-mediated unfaithful homeologous recombination repair (HomeoRR) in a dosage-dependent manner. BCR-ABL1 SH3 domain interacts with RAD51 proline-rich regions, resulting in direct phosphorylation of RAD51 on Y315 (pY315). RAD51(pY315) facilitates dissociation from the complex with BCR-ABL1 kinase, migrates to the nucleus, and enhances formation of the nuclear foci indicative of recombination sites. HomeoRR and RAD51 nuclear foci were strongly reduced by RAD51(Y315F) phosphorylation-less mutant. In addition, peptide aptamer mimicking RAD51(pY315) fragment, but not that with Y315F phosphorylation-less substitution, diminished RAD51 foci formation and inhibited HomeoRR in leukemia cells. In conclusion, we postulate that BCR-ABL1 kinase-mediated RAD51(pY315) promotes unfaithful HomeoRR in leukemia cells, which may contribute to accumulation of secondary chromosomal aberrations responsible for CML relapse and progression.

BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

BCR/ABL-transformed chronic myeloid leukemia (CML) cells accumulate numerous DNA double-strand breaks (DSB) induced by reactive oxygen species (ROS) and genotoxic agents. To repair these lesions BCR/ABL stimulate unfaithful DSB repair pathways, homologous recombination repair (HRR), nonhomologous end-joining (NHEJ), and single-strand annealing (SSA). Here, we show that BCR/ABL enhances the expression and increase nuclear localization of WRN (mutated in Werner syndrome), which is required for processing DSB ends during the repair. Other fusion tyrosine kinases (FTK), such as TEL/ABL, TEL/JAK2, TEL/PDGFβR, and NPM/ALK also elevate WRN. BCR/ABL induces WRN mRNA and protein expression in part by c-MYC-mediated activation of transcription and Bcl-xL-dependent inhibition of caspase-dependent cleavage, respectively. WRN is in complex with BCR/ABL resulting in WRN tyrosine phosphorylation and stimulation of its helicase and exonuclease activities. Activated WRN protects BCR/ABL-positive cells from the lethal effect of oxidative and genotoxic stresses, which causes DSBs. In addition, WRN promotes unfaithful recombination-dependent repair mechanisms HRR and SSA, and enhances the loss of DNA bases during NHEJ in leukemia cells. In summary, we postulate that BCR/ABL-mediated stimulation of WRN modulates the efficiency and fidelity of major DSB repair mechanisms to protect leukemia cells from apoptosis and to facilitate genomic instability.

Targeting Phosphotyrosine-315 In RAD51 Recombinase to Prevent BCR-ABL1 - Mediated Unfaithful Homeologous Recombination Repair.

AbstractAbstract 1190 Background:CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast phase (CML-BP) and cell lines transformed by non-mutated BCR-ABL1 kinase or tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) (Nowicki et al., Blood, 2006; Cramer et al., Cancer Res., 2008). DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS and radiation induced DSBs generated chromosomal aberrations in BCR-ABL1-positive leukemia cells (Koptyra et al., Leukemia, 2008), which may contribute to the malignant progression to CML-BP. We also showed that homologous recombination repair (HomoRR) driven by RAD51 recombinase is one of the major DSB repair mechanisms, which is stimulated by BCR-ABL1 (Slupianek et al., Molecular Cell, 2001). Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. Here we investigated if BCR-ABL1 can modulate RAD51 recombinase to corrupt the fidelity of recombination and if this process can be targeted to prevent genomic instability in leukemia cells. Result:To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease–inducible DSB site and a repair template displaying 1% sequence divergence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells, BCR-ABL1-32Dcl3 cells and BCR-ABL1(T315I)-32Dcl3 cells. BCR-ABL1 and BCR-ABL1(T315I) kinase caused about a 3-fold increase in HomeoRR activity implicating its role in the accumulation of chromosomal aberrations in CML cells. The magnitude of HomeoRR stimulation depended on BCR-ABL1 expression levels. RAD51 recombinase, a key regulator of recombination repair, forms a complex with BCR-ABL1 which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain of BCR-ABL1, but does not depend on its kinase activity. In fact, BCR-ABL1(K1172R) kinase-dead mutant formed complexes more abundantly with RAD51 than the kinase-active BCR-ABL1. BCR-ABL1-RAD51 complex formation resulted in direct phosphorylation of RAD51 on Y315 [RAD51(phosphoY315)], which is located in the vicinity of PP motifs in the C-terminal portion of RAD51. Phosphorylation-less C-terminal RAD51(Y315F) mutant formed a stronger complex with BCR-ABL1 than the wild-type form. Altogether, it appears that RAD51 PP bind to the SH3 domain of BCR-ABL1 kinase followed by quick phosphorylation of RAD51 on Y315 and disassembly of RAD51(phosphoY315) from the complex. Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. In fact RAD51(phosphoY315) was found in the nuclei of BCR-ABL1 leukemia cells, but not of parental cells and formed numerous foci on DSBs. Phosphorylation-less RAD51(Y315F) mutant abrogated foci formation and inhibited HomeoRR. Thus, BCR-ABL1 – RAD51(phosphoY315) pathway appears to promote unfaithful HomeoRR and chromosomal instability. To test if RAD51(phosphoY315) can be targeted to prevent unfaithful HomeoRR in leukemia cells, a peptide aptamer strategy was applied. Aptamer mimicking the RAD51(phosphoY315) fragment, but not that with the Y315F phosphorylation-less substitution inhibited RAD51 foci formation and HomeoRR activity in BCR-ABL1 leukemia cells. Conclusion:In summary, the BCR-ABL1-RAD51 axis may promote accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR-ABL1 kinase. We hypothesize that targeting BCR-ABL1-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BP progression. Furthermore, RAD51 and recombination is also affected by other oncogenic tyrosine kinases (OTKs) such as TEL-ABL1, TEL-PDGFR, ZNF198-FGFR1, TEL-JAK2, JAK2[V617F], NPM-ALK, IGF-1R, EGFR and FLT3-ITD suggesting that abrogation of OTK-mediated aberrant modulation of RAD51 may inhibit genetic instability and tumor progression. Disclosures:No relevant conflicts of interest to declare.

Targeting BCR/ABL-RAD51 Interaction to Prevent Unfaithful Homeologous Recombination Repair.

Abstract 3272Poster Board III-1CD34+ chronic myeloid leukemia (CML) stem/progenitor cells from chronic phase (CML-CP) and blast crisis (CML-BC) and cell lines transformed by non-mutated BCR/ABL kinase or the tyrosine kinase inhibitor (TKI)-resistant mutants contain numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) and genotoxic stress. In addition CD34+CD38- CML-CP and CD34+ CML-BC stem cell-enriched populations seem to display more DSBs than normal counterparts. DSBs may cause apoptosis if not repaired or chromosomal aberrations if repaired unfaithfully. We reported that numerous ROS- and radiation- induced DSBs induce chromosomal instability implicating enhanced, but unfaithful repair in BCR/ABL-positive leukemias [Leukemia, 2008]. We show here that BCR/ABL kinase (non-mutated and TKI-resistant mutants) facilitate recombination repair (RR) of DSBs. Although recombination usually represents a faithful mechanism of DSB repair, it may generate chromosomal aberrations when similar (homeologous), but not identical (homologous) templates are employed during the repair. To study unfaithful homeologous recombination repair (HomeoRR) a reporter repair cassette containing I-SceI endonuclease-;inducible DSB site and a repair template displaying 19% divergence sequence relative to the DSB site was integrated into the genome of 32Dcl3 murine hematopoietic cells and BCR/ABL-positive counterparts. BCR/ABL kinase caused about 3-fold increase in HomeoRR activity implicating its role in accumulation of chromosomal aberrations in CML cells. RAD51, a key regulator of recombination repair, forms a complex with BCR/ABL which depends on the proline- rich (PP) regions of RAD51 and the SH3 domain and SH2-catalytic domain (SH2-CD) linker of BCR/ABL. SH3+SH2-CD domains of BCR/ABL form a pocket binding the PP regions of RAD51. Single amino acid substitutions in the BCR/ABL SH3+SH2-CD pocket, which disrupted binding to the RAD51 PP regions reduced complex formation with RAD51. 32Dcl3 murine hematopoietic cells expressing BCR/ABL SH3+SH2-CD pocket mutant displayed slow proliferation rate and responded poorly to genotoxic stress despite intact kinase activity. On the other hand, disruption of the PP regions of RAD51 by P-L amino acid substitutions (PP-LL mutants) prevented its interaction with BCR/ABL SH3+SH2-CD pocket. Interestingly, expression of RAD51 PP-LL mutant abrogated the clonogenic capability of BCR/ABL-transformed leukemia cells, without any toxic effect on normal counterparts. BCR/ABL-RAD51 complex results in direct phosphorylation of RAD51 on Y315, which is located in the vicinity of PP motifs in the C-terminal portion (aa 271–339) of RAD51. C-terminal Y315F mutant formed more abundant complex with BCR/ABL that the wild-type form, but it did not restore the lost interaction of the PP/LL mutant. Thus, BCR/ABL-mediated RAD51[Y315] phosphorylation appears to be important for disassembly of RAD51 from BCR/ABL. In concordance, RAD51[Y315F] mutant remained mostly in the cytoplasm, while the wild-type protein accumulated in the nucleus in BCR/ABL-positive cells in response to DSBs induced by genotoxic treatment. In addition to the regulation of BCR/ABL-RAD51 interaction, phospho-Y315 is located in a critical fragment of RAD51 essential for its filament formation on DSBs, implicating its direct role in recombination. To test this hypothesis we employed a peptide aptamer strategy targeting phospho-Y315 of RAD51. Peptides corresponding to the RAD51 fragment containing phospho-Y315, but not these with Y315F substitution reduced HomeoRR activity by approximately 2-fold in BCR/ABL-positive leukemia cells. Altogether, it appears that PP-regions of RAD51 interact with SH3+SH2-CD niche of BCR/ABL, which leads to phosphorylation of RAD51 on Y315 and disassembly of the complex. Phospho-Y315 stimulates abundant nuclear localization of RAD51 on DSBs, which disrupts the mechanisms responsible for preventing recombination using divergent templates resulting in unfaithful HomeoRR in BCR/ABL-positive leukemia cells. In summary, BCR/ABL-RAD51 interaction promotes survival and accumulation of chromosomal aberrations of CML cells expressing non-mutated and TKI-resistant BCR/ABL kinase. We hypothesize that targeting BCR/ABL-RAD51 interaction may prevent/delay accumulation of secondary chromosomal aberrations and CML-BC progression. Disclosures:No relevant conflicts of interest to declare.

A High Throughput Genetic Screen Identifies New Early Meiotic Recombination Functions in Arabidopsis thaliana

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

Regulating the formation of DNA double-strand breaks in meiosis

During meiosis, cells deliberately form numerous DNA double-strand breaks (DSBs) in order to initiate homologous recombination, which exchanges genetic information between homologous chromosomes and promotes accurate chromosome segregation. DSB formation is restricted to a narrow window of time in meiosis, both for proper execution of the functions of recombination and to prevent production of toxic DNA lesions at inappropriate times. Two studies in this issue of Genes & Development provide important insight into the poorly understood mechanisms that ensure proper timing of DSB formation.

The multiple roles of the Mre11 complex for meiotic recombination

During the first meiotic prophase, numerous DNA double-strand breaks (DSB) are formed in the genome in order to initiate recombination between homologous chromosomes. The conserved Mre11 complex, formed of Mre11, Rad50 and Nbs1 (Xrs2 in Saccharomyces cerevisiae) proteins, plays a crucial role in mitotic cells for sensing and repairing DSB. In meiosis the Mre11 complex is also required for meiotic recombination. Depending on the organisms, the Mre11 complex is required for the formation of the DSB catalysed by the transesterase Spo11 protein. It then plays a unique function in removing covalently attached Spo11 from the 5' extremity of the breaks through its nuclease activity, to allow further break resection. Finally, the Mre11 complex also plays a role during meiosis in bridging DNA molecules together and in sensing Spo11 DSB and activating the DNA damage checkpoint. In this article the different biochemical functions of the Mre11 complex required during meiosis are reviewed, as well as the consequences of Mre11 complex inactivation for meiosis in several organisms. Finally, I describe the meiotic phenotypes of several animal models that have been developed to model hypomorphic mutations of the Mre11 complex, involved in humans in some genetic instability disorders.

Genetic variance modifies apoptosis susceptibility in mature oocytes via alterations in DNA repair capacity and mitochondrial ultrastructure

Although the identification of specific genes that regulate apoptosis has been a topic of intense study, little is known of the role that background genetic variance plays in modulating cell death. Using germ cells from inbred mouse strains, we found that apoptosis in mature (metaphase II) oocytes is affected by genetic background through at least two different mechanisms. The first, manifested in AKR/J mice, results in genomic instability. This is reflected by numerous DNA double-strand breaks in freshly isolated oocytes, causing a high apoptosis susceptibility and impaired embryonic development following fertilization. Microinjection of Rad51 reduces DNA damage, suppresses apoptosis and improves embryonic development. The second, manifested in FVB mice, results in dramatic dimorphisms in mitochondrial ultrastructure. This is correlated with cytochrome c release and a high apoptosis susceptibility, the latter of which is suppressed by pyruvate treatment, Smac/DIABLO deficiency, or microinjection of 'normal' mitochondria. Therefore, background genetic variance can profoundly affect apoptosis in female germ cells by disrupting both genomic DNA and mitochondrial integrity.

Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells.

Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.