Induction Of Homologous Recombination Research Articles

Maternal exposure to dibutyl phthalate regulates MSH6 crotonylation to impair homologous recombination in fetal oocytes

Homologous recombination (HR) during early oogenesis repairs programmed double-strand breaks (DSBs) to ensure female fertility and offspring health. The exposure of fetal ovaries to endocrine disrupting chemicals (EDCs) can cause reproductive disorders in the adulthood. The EDC dibutyl phthalate (DBP) is widely distributed in flexible plastic products, leading to ubiquitous human exposure. Here, we report that maternal exposure to DBP caused gross aberrations in meiotic prophase I of fetal oocytes, including delayed progression, impaired DNA damage response, uncoupled localization of DMC1 and RAD51, and decreased HR. However, programmed DSBs were efficiently repaired. DBP exposure negatively regulated lysine crotonylation (Kcr) of MSH6. Similar meiotic defects were observed in fetal ovaries with targeted disruption of Msh6, and mutation of K544cr of MSH6 impaired its association with Ku70, thereby promoting non-homologous end joining (NHEJ) and inhibiting HR. Unlike mature F1 females, F2 female mice exhibited premature follicular activation, precocious puberty, and anxiety-like behaviors. Therefore, DBP can influence early meiotic events, and Kcr of MSH6 may regulate preferential induction of HR or NHEJ for DNA repair during meiosis.

Abstract 1742: Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases

Abstract Type II topoisomerases play key roles in transcription, replication and chromosome segregation. These enzymes modulate DNA structure by generating transient DNA breaks where the protein is covalently bound to DNA. Anti-cancer drugs targeting topoisomerase II (Top2) inhibit the religation of enzyme-induced breaks, and exert their anti-cancer effects through the generation of enzyme mediated DNA damage. In addition to small molecule inhibitors, it has been suggested that aberrant DNA structures or DNA lesions may also lead to Top2/DNA lesions. A mammalian Top2 isoform, Top2β has been suggested to play a key role in hormone-mediated transcription, and in carrying out this function, generates potentially oncogenic DNA damage. We have assessed the generation of potentially oncogenic DNA damage by Top2β in model organisms, and using the purified protein. We found that ectopic expression of Top2β is poorly tolerated in repair deficient yeast strains, while expression of the mammalian Top2α isoform lacks this property. We measured the induction of homologous recombination by Top2β in yeast and found that ectopic expression of the enzyme led to elevated levels of recombination. Recently, a de novo mutation in Top2β was reported in a patient with severe developmental delay resulting from an His63Tyr substitution in Top2β 1. We found that Top2β (His63Tyr) could not complement a defect in yeast Top2, but encoded an enzyme that was catalytically active, and induced elevated levels of recombination. Because of the inferred location of His63 in the ATPase domain of Top2β, and proximity to the ATP binding site of the enzyme, we hypothesized that the mutant protein was partially defective in ATP regulated DNA cleavage. To further explore how ATP regulates Top2β cleavage, we examined the effects of expressing a Top2β variant completely lacking the N-terminal ATPase domain of Top2β. While this mutant did not complement yeast top2 mutants, we found that it s expression induced 10-20 fold higher levels of recombination than cells expressing wild type Top2β. This result indicates that an ATP deficient variant of Top2β induces very high levels of DNA damage. We suggest that regulation of the ATPase activity of Top2β may be critical for maintaining genome stability, and may be responsible for induction of DNA damage during hormone-mediated transcription. 1. C.W. Lam et al., Global developmental delay and intellectual disability associated with a de novo TOP2B mutation. Clinica Chimica Acta 469:63-68, 2017. Citation Format: Amanda M. Johnson, Lokha Ranjani A. Boopathy, Raveena Gupta, Hannah N. Miles, Matthew Gilbertson, Karin C. Nitiss, John L. Nitiss. Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1742.

Genotoxicity of Zinc Oxide Nanoparticles in Plants Demonstrated Using Transgenic Arabidopsis thaliana.

As important members of earth biosphere, higher plants are inevitably exposed to nanoparticles (NP) released into the environment. Therefore, determining NP-induced phytotoxicity is ecologically important. Currently, researches into genotoxic effects of NP on plants are limited. In this study, Arabidopsis thaliana lines transgenic for homologous recombination (HR) and transcriptional gene silencing (TGS) reporter genes were for the first time adopted to assess the genotoxicity of Zinc oxide NP (ZnO-NP). Results showed that the root exposure to ZnO-NP led to increased HR and alleviation of TGS in the aerial tissues, indicative of the genotoxicity of ZnO-NP in plants. The increased Zn content after root exposure to ZnO-NP and the similar induction of HR and TGS alleviation after root exposure to equivalent Zn ions suggested that the genotoxicity of ZnO-NP might be mainly induced by Zn ions in aerial tissues that were transported from decomposed ZnO-NP in either medium or plant roots.

Prostate Cancer Patients with Late Radiation Toxicity Exhibit Reduced Expression of Genes Involved in DNA Double-Strand Break Repair and Homologous Recombination.

Severe late damage to normal tissue is a major limitation of cancer radiotherapy in prostate cancer patients. In a recent retrospective study, late radiation toxicity was found to relate to a decreased decay of γ-H2AX foci and reduced induction of DNA double-strand break repair genes. Here, we report evidence of prognostic utility in prostate cancer for γ-H2AX foci decay ratios and gene expression profiles derived from ex vivo-irradiated patient lymphocytes. Patients were followed ≥2 years after radiotherapy. Clinical characteristics were assembled, and toxicity was recorded using the Common Terminology Criteria (CTCAE) v4.0. No clinical factor was correlated with late radiation toxicity. The γ-H2AX foci decay ratio correlated negatively with toxicity grade, with a significant difference between grade ≥3 and grade 0 patients (P = 0.02). A threshold foci decay ratio, determined in our retrospective study, correctly classified 23 of 28 patients with grade ≥3 toxicity (sensitivity 82%) and 9 of 14 patients with grade 0 toxicity (specificity 64%). Induction of homologous recombination (HR) repair genes was reduced with increasing toxicity grade. The difference in fold induction of the HR gene set was most pronounced between grade 0 and grade ≥3 toxicity (P = 0.008). Notably, reduced responsiveness of HR repair genes to irradiation and inefficient double-strand break repair correlated with severe late radiation toxicity. Using a decay ratio classifier, we correctly classified 82% of patients with grade ≥3 toxicity, suggesting a prognostic biomarker for cancer patients with a genetically enhanced risk for late radiation toxicity to normal tissues after radiotherapy. Cancer Res; 77(6); 1485-91. ©2017 AACR.

ABL Tyrosine Kinase Plays an Important Role in Mechanisms Involved in Genomic Instability in Multiple Myeloma

Homologous recombination (HR) is a DNA repair mechanism that uses extensive sequence homology in the participating DNA molecules for an accurate repair. In a normal cellular environment, HR is the most precise DNA repair mechanism and therefore has a unique role in the maintenance of genomic integrity and stability. Normally HR is tightly regulated, however, as it involves incision and recombination of genomic DNA fragments, if dysregulated or dysfunctional, it can also be deleterious. Consistent with this view, we have shown that elevated HR activity mediates genomic instability and development of drug resistance in MM. Here we have now investigated the mechanism that may contribute to dysregulation of HR and genomic instability in MM, as well as evaluated an agent able to decrease acquisition of new genomic changes. It has been shown that Abl kinase regulates recombinase RAD51 by affecting its expression, stability as well as phosphorylation at Y315. Phosphorylation of RAD51 (at Y315) mediates its dissociation from BCR-ABL1 kinase and migration to the nucleus to form nuclear foci, one of the initial steps in HR. We have evaluated nilotinib, a small molecule inhibitor of Abl kinase and observed that it inhibited HR activity in all MM cell lines tested, in a dose-dependent manner. At 5 µM, nilotinib inhibited HR activity in MM1S, RPMI 8226 and U266 MM cells by 64%, 78% and 80%, respectively. Nilotinib led to reduced phosphorylation of RAD51 at Y315, the phosphorylation which affects RAD51 migration. Nilotinib-mediated inhibition of RAD51 and HR activity was also associated with reduced DNA breaks, as indicated by reduced levels of g-H2AX. To determine the impact of nilotinib on genome stability, MM (RPMI 8226) cells were cultured in the presence of nilotinib for three weeks and the impact of this treatment on appearance of new copy number changes was evaluated using SNP arrays. Using day 0 cells as baseline to identify new copy number events at 3 weeks, the acquisition of new genomic changes was inhibited by 50% in the presence of nilotinib. As we have previously reported that induction of HR helps develop dexamethasone resistance in a short period of time, we investigated whether inhibition of HR by nilotinib may improve efficacy of melphalan and dexamethasone in MM. Nilotinib (at 2.5 µM) significantly increased the efficacy of melphalan (10 µM); and dexamethasone (10 nM) in RPMI 8226 cells. The relation between these observed effects and inhibition of HR is being investigated.In conclusion, we have observed that Abl tyrosine kinase plays an important role in genomic instability in myeloma and its inhibition using nilotinib, suppresses the underlying mechanism of genomic instability and reduces acquisition of new genomic changes with potential for clinical application. DisclosuresNo relevant conflicts of interest to declare.

Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

Abstract C52: Induction of homologous recombination by DNA nicks

Abstract Homologous recombination (HR) is a major mechanism of DNA repair, such that mutations in HR genes are associated with tumor susceptibility. We have used a recently adapted system for genome engineering to study HR mechanisms in mammalian cells. Clustered regularly-interspaced short palindromic repeats (CRISPR) are prokaryotic adaptive immune systems that specifically cleave nucleic acids of invading pathogens. The effector phase of CRISPR system in Staphylococcus pyogenes consists of two main components, the endonuclease Cas9 and RNA strands that dictate the recognition sequence cleaved by Cas9. The activity of Cas9 resides in 2 nucleolytic domains, HNH-like and RuvC1-like, such that mutating the active site of either of these domains turns Cas9 into a DNA nicking enzyme. Here we applied the commonly used DR-GFP HR reporter to investigate the repair of both DNA double-strand breaks (DSBs) and nicks induced by Cas9 in human cells. We show that a DSB, introduced by wild-type (wt) Cas9 and a guide RNA (gRNA) directed to the I-SceI site of the DR-GFP reporter, induces HR at levels similar to the I-SceI endonuclease, demonstrating the robustness of the system and confirming the potential of the CRISPR system as a genome engineering tool. Cas9 with mutations in either of the nucleolytic domains also stimulates HR, albeit at much lower levels, suggesting that a nick can also be repaired by HR. Stimulation of HR by DNA nicks is not reduced by PARP inhibition and has the same genetic requirements as DSB-induced HR. Given that SSBs are induced by endogenous DNA metabolic processes as well as by many anti-cancer treatments, our results may advance the understanding of mechanisms responsible for repair of these abundant DNA lesions. Citation Format: Lianne E.M. Vriend, Przemek M. Krawczyk, Maria Jasin. Induction of homologous recombination by DNA nicks. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C52.

Induction of homologous recombination following in utero exposure to DNA-damaging agents

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures.

NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes.

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP–BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP–BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

The Induction of Bystander Mutagenic EffectsIn Vivoby Alpha-Particle Irradiation in WholeArabidopsis thalianaPlants

Our previous studies demonstrated distant/abscopal bystander effects in A. thaliana seeds and embryos; the postembryonic development of bystander tissues, such as root hair differentiation, primary root elongation, lateral root initiation and survival, were inhibited significantly by localized irradiation with microbeam protons and low-energy ions. In the present study, we further investigated radiation-induced bystander mutagenic effects in vivo in A. thaliana plants using homologous recombination (HR) and the expression level of the HR-related AtRAD54 gene as mutagenic end points. We found that α-particle irradiation of distal primary roots of young seedlings resulted in a significant increase in the frequency of HR in the aerial plants; the increased induction of HR occurred in every true leaf over the course of rosette development. Moreover, we also found that localized α-particle irradiation of roots induced a short-term up-regulated expression of the HR-related AtRAD54 gene in the nonirradiated aerial plants. These results suggested the existence of bystander mutagenic effects in vivo in plants. Treatment with the ROS scavenger DMSO dramatically reduced the effects of localized root irradiation on the induction of HR and expression of the AtRAD54 gene in bystander tissues, suggesting that ROS play a critical role in mediating the bystander mutagenic effects in plants.

The Induction of Bystander Mutagenic Effects In Vivo by Alpha-Particle Irradiation in Whole Arabidopsis thaliana Plants

Our previous studies demonstrated distant/abscopal bystander effects in A. thaliana seeds and embryos; the postembryonic development of bystander tissues, such as root hair differentiation, primary root elongation, lateral root initiation and survival, were inhibited significantly by localized irradiation with microbeam protons and low-energy ions. In the present study, we further investigated radiation-induced bystander mutagenic effects in vivo in A. thaliana plants using homologous recombination (HR) and the expression level of the HR-related AtRAD54 gene as mutagenic end points. We found that alpha-particle irradiation of distal primary roots of young seedlings resulted in a significant increase in the frequency of HR in the aerial plants; the increased induction of HR occurred in every true leaf over the course of rosette development. Moreover, we also found that localized alpha-particle irradiation of roots induced a short-term up-regulated expression of the HR-related AtRAD54 gene in the nonirradiated aerial plants. These results suggested the existence of bystander mutagenic effects in vivo in plants. Treatment with the ROS scavenger DMSO dramatically reduced the effects of localized root irradiation on the induction of HR and expression of the AtRAD54 gene in bystander tissues, suggesting that ROS play a critical role in mediating the bystander mutagenic effects in plants.

Genotoxicity/mutagenicity of formaldehyde revealed by the Arabidopsis thaliana plants transgenic for homologous recombination substrates

Formaldehyde (FA) is a major industrial chemical and has been extensively used in the manufacture of synthetic resins and chemicals. The use of FA-containing industrial materials in daily life exposes human to FA extensively. Numerous studies indicate that FA is genotoxic, and can induce various genotoxic effects in vitro and in vivo. The primary DNA lesions induced by FA are DNA–protein crosslinks (DPCs). Recently, it has been reported that the homologous recombination (HR) mechanism is involved in the repair of DPCs, suggesting the homologous recombination could be a potential indicator for the genotoxicity/mutagenicity of FA. However, it has not yet been reported that organisms harboring recombination substrates are used for the detection of genotoxic/mutagenic effects of FA. In this present study, an Arabidopsis thaliana-line transgenic for GUS recombination substrates was used to study the genotoxicity/mutagenicity of FA, and the results showed that FA-exposure significantly increased the induction of HR in growing plants, but not in dormant seeds. We also observed an early up-regulation of expression of HR-related gene, AtRAD54, after FA-exposure. Moreover, the pretreatment with glutathione (GSH) suppressed drastically the induction of HR by FA-exposure.

Irradiated Esophageal Cells are Protected from Radiation-Induced Recombination by MnSOD Gene Therapy

Radiation-induced DNA damage is a precursor to mutagenesis and cytotoxicity. During radiotherapy, exposure of healthy tissues can lead to severe side effects. We explored the potential of mitochondrial SOD (MnSOD) gene therapy to protect esophageal, pancreatic and bone marrow cells from radiation-induced genomic instability. Specifically, we measured the frequency of homologous recombination (HR) at an integrated transgene in the Fluorescent Yellow Direct Repeat (FYDR) mice, in which an HR event can give rise to a fluorescent signal. Mitochondrial SOD plasmid/liposome complex (MnSOD-PL) was administered to esophageal cells 24 h prior to 29 Gy upper-body irradiation. Single cell suspensions from FYDR, positive control FYDR-REC, and negative control C57BL/6NHsd (wild-type) mouse esophagus, pancreas and bone marrow were evaluated by flow cytometry. Radiation induced a statistically significant increase in HR 7 days after irradiation compared to unirradiated FYDR mice. MnSOD-PL significantly reduced the induction of HR by radiation at day 7 and also reduced the level of HR in the pancreas. Irradiation of the femur and tibial marrow with 8 Gy also induced a significant increase in HR at 7 days. Radioprotection by intraesophageal administration of MnSOD-PL was correlated with a reduced level of radiation-induced HR in esophageal cells. These results demonstrate the efficacy of MnSOD-PL for suppressing radiation-induced HR in vivo.

Arabidopsis SNI1 and RAD51D regulate both gene transcription and DNA recombination during the defense response

The plant immune response known as systemic acquired resistance (SAR) is a general defense mechanism that confers long-lasting resistance against a broad spectrum of pathogens. SAR triggers many molecular changes including accumulation of antimicrobial pathogenesis-related (PR) proteins. Transcription of PR genes in Arabidopsis is regulated by the coactivator NPR1 and the repressor SNI1. Pathogen infection also triggers an increase in somatic DNA recombination, which results in transmission of changes to the offspring of infected plants. However, it is not known how the induction of homologous recombination during SAR is controlled. Here, we show that SNI1 and RAD51D regulate both gene expression and DNA recombination. In a genetic screen for suppressors of sni1, we discovered that RAD51D is required for NPR1-independent PR gene expression. As a result, the rad51d mutant has enhanced disease susceptibility. Besides altered PR gene expression, rad51d plants are hypersensitive to DNA-damaging agents and are impaired in homologous recombination. The dual role of RAD51D and SNI1 in PR gene transcription and DNA recombination suggests a mechanistic link between the short-term defense response and a long-term survival strategy.

Critical Role of Recombinase (HsRAD51) in Genetic Instability in Multiple Myeloma.

A number of cancers, including multiple myeloma (MM), show a significant genetic instability that evolves with the progression of disease. The molecular basis for the generation of genetic diversity in cancer cells has therefore emerged as an important focus of investigation. We have previously shown that homologous recombination (HR), required for a variety of normal cellular processes, is dysregulated in multiple myeloma and leads to a progressive accumulation of genetic variation over time. Moreover the inhibition or stimulation of HR activity in MM cells, by altering components of the HR pathway, concordantly affects the acquisition of new genetic changes. Consistent with this, an elevated frequency of cancer is observed in patients whose cells have elevated rate of recombination. HsRAD51 recombinase, is a key molecule in HR pathway and its dysregulated expression can increase in HR activity, leading to increased rate of mutations and development or progression of cancer. Based on these data, we hypothesized that hsRAD51 promoter activity may be a target for carcinogenic agents and ability to measure the hsRAD51 promoter activity may provide a simpler method to assess oncogenic potential of test agents. To this end we constructed a mammalian expression vector containing firefly luciferase gene downstream of hsRAD51 promoter. This plasmid was transfected into normal human plasma cells, multiple myeloma cell lines (ARP, OPM1, U266), and normal human diploid fibroblasts. The cells were allowed to recover for 24 hrs and then treated with agents with carcinogenic or anti-oxidant activities, or control vehicles for 2 hrs; lysed and luciferase activity assessed. RAD51 promoter activity was elevated 7- to 1000-fold in MM cells relative to normal plasma cells. Exposure of MM cells to a carcinogen (nickel chloride) was able to further induce hsRAD51 promoter activity (2-fold) in MM cells with already over-expression of hsRAD51. However, the anti-oxidant tocopherol and cannabinol were able to reduce hsRAD51 promoter activity by 3- and 8-fold respectively, while vitamin C and Selenium had minimal effects. In normal diploid fibroblasts, although the exposure to a non-carcinogen, Sodium azide did not induce hsRAD51 promoter activity, the exposure to agents with known transforming ability, Mitomycin C and Nickel chloride, led to a significant induction of hsRAD51 promoter activity (2.5 and 3.5-fold respectively). Moreover, the ability of these chemicals to induce RAD51 promoter activity covaried with their ability to induce homologous recombination activity as measured by a novel plasmid based assay (r = 0.9). We also demonstrate that Induction of RAD51 and HR or its inhibition using siRNAs targeting hsRAD51 can inhibit acquisition of new mutations in MM cells as measured by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. These preliminary results provides us with a simple and powerful method to measure oncogenic potential of known carcinogens and demonstrate a critical role for RAD51 in ongoing genomic instability in MM and a rationale to target RAD51 to prevent progression.

1056. Suppression and Enhancement of Gene Repair by the Modulation of ATM Function in Mammalian Cells

Targeted gene repair utilizes oligonucleotides to direct single base alterations in the genomes of mammalian cells. These vectors are designed to hybridize to the site of interest and to create a single mismatch with the mutant base. Although the mechanism of the nucleotide exchange phase of the reaction remains to be fully elucidated, evidence suggests that the induction of homologous recombination (HR) in response to DNA damage elevates DNA pairing activity and results in increased levels of gene repair. The HR pathway is controlled by the activation of the cell cycle checkpoint regulator, Ataxia-Telangiectasia mutated (ATM), which is induced by either DNA damage or by the presence of single stranded DNA. The radio-sensitizer, caffeine, has previously been shown to inhibit ATM kinase activity, by interfering with the phosphorylation of downstream substrates. When caffeine was added to a gene repair reaction prior to the introduction of the oligonucleotide, the frequency of correction was reduced, reinforcing the importance of HR in gene repair. If, however, caffeine was added after the oligonucleotide, the correction frequency was enhanced suggesting a dual functionality for caffeine in the gene repair reaction. Thus, we utilize caffeine as a reagent and a tool to understand the regulation of this reaction in greater detail. ATM activation in cells exposed to 4mM caffeine was examined following oligonucleotide delivery. At 18 and 24 hours, the level of ATM activation was increased significantly and even at 48 hours post-delivery, when ATM activation form the introduction of the ssDNA vector disappears, activated ATM was still quite discernable in caffeine treated cells. This sustained ATM activation could be explained by a concurrent effect of caffeine on the gene repair reaction; the oligonucleotide is retained by the cell for a longer period than in untreated cells. At 16 hours post electroporation, both cell populations exhibited about a 70% uptake of the oligonucleotide (as measured by the fluorescence of a FAM conjugated oligonucleotide). However, between 16 and 48 hours, the non-treated cells exhibit a steep drop in oligonucleotide levels, as compared to the caffeine treated cells. This suggests that the oligonucleotide activates ATM in all cells but in cells treated with caffeine, it reactivates ATM through an extended cellular half-life. The use of caffeine as a tool for studying the regulatory pathways of gene repair lead us to conclude that the activation of ATM is a critical step in promoting successful gene correction. On one hand ATM is required to induce HR which catalyses the first phase of gene repair (DNA pairing), but on the other hand, activated ATM can block the replication of corrected cells affecting the overall frequency of the reaction. We are now modulating the levels of ATM so that the balance can be tipped in favor of more robust levels of gene repair.

Molecular Mechanisms Underlying the Development of Drug Resistance in Multiple Myeloma.

A prominent feature of most cancers including multiple myeloma (MM) is a striking genetic instability, leading to ongoing accrual of mutational changes some of which underlie tumor progression, including development of drug resistance and metastasis. The molecular basis for the generation of genetic diversity in cancer cells has thus emerged as an important focus of investigation and a target for successful eradication. We have previously observed that homologous recombination (HR) is upregulated in MM. Utilizing a genomewide LOH assay based on SNP genotyping (Affymetrix) as a tool to estimate the rate of mutation and genomic instability, we have observed that over time elevated HR leads to progressive accumulation of genetic variation in MM cell lines and patient cells; and inhibition of HR activity in MM cells by altering components of the HR pathway concordantly affects the acquisition of new genetic changes. As HR activity is dependent on concerted action of number of genes, instead of over expressing single HR related gene, we utilized nickel chloride, a known recombinogen to evaluate effects of increased HR activity on the development of genomic diversity. We cultured ARP cells in the presence or absence of nickel chloride, over a period of 90 days. Genome-wide LOH was evaluated by comparing genotypes before and after the 90-day interval. In three independent experiments treatment of cells with nickel chloride increased the number of new LOH sites by more than 12-fold. We next evaluated the effect of induction of HR and the consequent increase in genetic aberrations, on development of drug resistance in MM. Myeloma cells were cultured with nickel chloride as a potent inducer of HR and dexamethasone (10−8M); control cells were exposed to dexamethasone alone. The cell viability was measured weekly. No live cells were detected in cultures exposed to dexamethasone alone; while >95% cells exposed to both nickel chloride and dexamethasone were alive following 2 weeks culture. These findings were confirmed by 3 independent experiments. The development of drug resistance was further confirmed by demonstrating no significant effects of dexamethasone on these cells at 10−6M concentrations for >1 week. Dexamethasone at this concentration kills all control cells by day 3. Evaluation of development of resistance to other agents is underway. We propose that continued accumulation of new genetic changes mediated by HR, as demonstrated here, provides the molecular events required to develop drug resistance; and its inhibition may allow us to successfully treat MM cells without the currently observed development of resistance. HR may be a potential therapeutic target to maintain chemo sensitivity of the tumors.

The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination.

Rad17 is involved in DNA checkpoint control in yeast and human cells. A homologue of this gene as well as other genes of the pathway (the 9-1-1 complex) are present in Arabidopsis and share conserved sequence domains with their yeast and human counterparts. DNA-damaging agents induce AtRAD17 transcriptionally. AtRAD17 mutants show increased sensitivity to the DNA-damaging chemicals bleomycin and mitomycin C (MMC), which can be reversed by complementation, suggesting that the loss of function of Rad17 disturbs DNA repair in plant cells. Our results are further confirmed by the phenotype of a mutant of the 9-1-1 complex (Rad9), which is also sensitive to the same chemicals. AtRAD9 and AtRAD17 seem to be epistatic as the double mutant is not more sensitive to the chemicals than the single mutants. The mutants show a delay in the general repair of double-strand breaks (DSBs). However, frequencies of intrachromosomal homologous recombination (HR) are enhanced. Nevertheless, the mutants are proficient for a further induction of HR by genotoxic stresses. Our results indicate that a mutant Rad17 pathway is associated with a general deregulation of DNA repair, which seems to be correlated with a deficiency in non-homologous DSB repair.

Systemic plant signal triggers genome instability.

Previously, we have shown that infection of tobacco plants with a viral pathogen triggers local and systemic induction of homologous recombination (HR). Here, we have tested the hypothesis of whether free radicals are potentially involved in the induction of the systemic effect. We report a significant induction of HR in tobacco plants treated with radical-generating agents, UVC or rose Bengal (RB). Importantly, the recombination increase was observed in local (treated) as well as systemic (non-treated) tissue. The systemic increase in recombination implies the existence of a signal that is transmitted to non-treated tissue. Several sets of grafting experiments proved the generation of said signal by both RB and UVC exposure. A statistically significant increase in HR was observed in tissue that received a systemic signal via a grafted leaf. Similar data were obtained from transgenic plants naphthalene degrading salicylate 1-hydroxylase (NahG) unable to accumulate salicylic acid (SA). Interestingly, pre-treatment of plants with the radical-scavenging compound N-acetyl-l-cysteine (NAC) led to a significantly lower recombination increase upon grafting after treatment with UVC and RB. Moreover, leaves taken for grafting from NAC-pre-treated plants exhibited a lower level of oxidized organic compounds. Our data suggest the involvement of free radical production in either generation or maintenance of the recombination signal. We discuss potential mechanisms for generation of the signal and possible adaptive advantages of enhanced genomic flexibility following exposure to DNA-damaging agents.

UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation.

Plants are continuously subjected to UV-B radiation (UV-B; 280-320 nm) as a component of sunlight causing damage to the genome. For elimination of DNA damage, a set of repair mechanisms, mainly photoreactivation, excision, and recombination repair, has evolved. Whereas photoreactivation and excision repair have been intensely studied during the last few years, recombination repair, its regulation, and its interrelationship with photoreactivation in response to UV-B-induced DNA damage is still poorly understood. In this study, we analyzed somatic homologous recombination in a transgenic Arabidopsis line carrying a beta-glucuronidase gene as a recombination marker and in offsprings of crosses of this line with a photolyase deficient uvr2-1 mutant. UV-B radiation stimulated recombination frequencies in a dose-dependent manner correlating linearly with cyclobutane pyrimidine dimer (CPD) levels. Genetic deficiency for CPD-specific photoreactivation resulted in a drastic increase of recombination events, indicating that homologous recombination might be directly involved in eliminating CPD damage. UV-B irradiation stimulated recombination mainly in the presence of photosynthetic active radiation (400-700 nm) irrespective of photolyase activities. Our results suggest that UV-B-induced recombination processes may depend on energy supply derived from photosynthesis.