DNA Double-strand Break Repair Machinery Research Articles

DNA double-strand break repair machinery in Penaeid crustaceans: A focus on the Non-Homologous End-Joining pathway.

DNA double-strand breaks (DSBs) are repaired through three major pathways: Non-Homologous End-Joining (NHEJ), Microhomology-Mediated End-Joining (MMEJ), and Homology-Directed Repair (HDR), each requiring a specific set of diverse proteins. Such pathways and their proteins have been studied in model organisms, including arthropods; however, DSB repair pathways are scarcely described in Crustacea, a taxon that includes the commercially valuable penaeid shrimps (Crustacea: Decapoda: Penaeidae). In this work, transcriptome and proteome databases of Penaeus vannamei and other Crustacea species were scrutinized for each protein of the NHEJ pathway. The structural and functional attributes of such proteins in penaeids were determined using bioinformatics. Additionally, the expression of the NHEJ-related Ku70, Ku80, DNA-PKcs, DNA ligase 4 (Lig4), and HDR- and MMEJ-related protein transcripts were assessed in P. vannamei gills, midgut gland, hemocytes, and muscle by RT-PCR. DSB repair protein transcripts were found expressed in the four assayed tissues, particularly in the gills and midgut gland. Among DSB repair proteins, all the analyzed transcripts of proteins related to the NHEJ pathway were present in gills. To the best of our knowledge, this is the first report on the expression of DSB repair proteins in Decapoda. Together, proteomic, transcriptomic, and expression data suggest the functionality of NHEJ, HDR, and MMEJ pathways in P. vannamei and other decapods. The information presented here contributes to understanding the response to DSB breaks in shrimps, describing possible outcomes in oxidative stress studies and also in the designing of gene editing strategies, which have not been developed in Penaeidae.

Immediate-Early, Early, and Late Responses to DNA Double Stranded Breaks.

Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called ‘fast-kinetic’ canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either ‘slow-kinetic’ cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself.

Microcephaly family protein MCPH1 stabilizes RAD51 filaments.

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2–RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51–ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.

Efficient Nuclease-Directed Integration of Lentivirus Vectors into the Human Ribosomal DNA Locus

Lentivirus vectors (LVs) are efficient tools for gene transfer, but the non-specific nature of transgene integration by the viral integration machinery carries an inherent risk for genotoxicity. We modified the integration machinery of LVs and harnessed the cellular DNA double-strand break repair machinery to integrate transgenes into ribosomal DNA, a promising genomic safe-harbor site for transgenes. LVs carrying modified I-PpoI-derived homing endonuclease proteins were characterized in detail, and we found that at least 21% of all integration sites localized to ribosomal DNA when LV transduction was coupled to target DNA cleavage. In addition to the primary sequence recognized by the endonuclease, integration was also enriched in chromatin domains topologically associated with nucleoli, which contain the targeted ribosome RNA genes. Targeting of this highly repetitive region for integration was not associated with detectable DNA deletions or negative impacts on cell health in transduced primary human Tcells. The modified LVs characterized here have an overall lower risk for insertional mutagenesis than regular LVs and can thus improve the safety of gene and cellular therapy.

Pro-oncogenic action of LOX-1 and its splice variant LOX-1\u03944 in breast cancer phenotypes

The identification of new predictive biomarkers and therapeutic target for tailored therapy in breast cancer onset and progression is an interesting challenge. OLR-1 gene encodes the cell membrane receptor LOX-1 (lectin-like oxidized low-density lipoprotein receptor). We have recently identified a novel alternative OLR-1 isoform, LOX-1Δ4, whose expression and functions are still not clarified. In the present paper, we demonstrated that LOX-1 is overexpressed in 70% of human breast cancer (n = 47) and positively correlated to the tumor stage and grade (p < 0.01). Observations on LOX-1 and its splice variant Δ4 pointed out a different expression pattern correlated to breast cancer phenotypes. Overexpressing LOX-1 and LOX-1Δ4 in vitro, we obtained a strong enhancement of proliferative rate and a downregulation of cell death-related proteins. In addition, we observed a strong modulation of histone H4 acetylation and Ku70, the limiting factor of DNA double-strand breaks repair machinery implied in apoptosis inhibition and drug resistance acquisition. Moreover, LOX-1Δ4 overexpression is able to increase proliferation in a non-tumorigenic epithelial cell line, MCF12-F, acting as an oncogene. Altogether, these results suggest that LOX-1 may acts as a molecular link among metabolism, inflammation and cancer, indicating its potential role as biomarker and new molecular target, representing an attractive and concrete opportunity to improve current strategies for breast cancer tailored therapy.

Intrinsic LINE-1 Hypomethylation and Decreased Brca1 Expression are Associated with DNA Repair Delay in Irradiated Thyroid Cells.

Exposure to ionizing radiation greatly increases the risk of developing papillary thyroid carcinoma (PTC), especially during childhood, mainly due to gradual inactivation of DNA repair genes and DNA damages. Recent molecular characterization of PTC revealed DNA methylation deregulation of several promoters of DNA repair genes. Thus, epigenetic silencing might be a plausible mechanism for the activity loss of tumor suppressor genes in radiation-induced thyroid tumors. Herein, we investigated the impact of ionizing radiation on global methylation and CpG islands within promoter regions of homologous recombination (HR) and non-homologous end joining (NHEJ) genes, as well as its effects on gene expression, using two well-established normal differentiated thyroid cell lines (FRTL5 and PCCL3). Our data reveal that X-ray exposure promoted G2/M arrest in normal thyroid cell lines. The FRTL5 cells displayed a slower kinetics of double-strand breaks (DSB) repair and a lower long interspersed nuclear element-1 (LINE-1) methylation than the PCCL3 cells. Nevertheless, acute X-ray exposure does not alter the expression of genes involved in HR and NHEJ pathways, apart from the downregulation of Brca1 in thyroid cells. On the other hand, HR and NHEJ gene expressions were upregulated in radiation-induced senescent thyroid cells. Taken together, these data suggest that FRTL5 cells intrinsically have less efficient DNA DSB repair machinery than PCCL3 cells, as well as genomic instability, which could predispose the FRTL5 cells to unrepaired DSB lesions and, therefore, gene mutations.

Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage.

Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa.

Genome Editing: A New Approach to Human Therapeutics.

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1.

TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.

DNA repair inhibition by DT01 as an adjuvant therapy at each stage of hepatocellular cancer (HCC) treatment.

303 Background: Hepatocellular carcinoma (HCC) is the most common liver cancer. Radiofrequency ablation (RFA), transarterial chemoembolization (TACE) or chemotherapy (CT) can be considered at each stage of HCC. The efficacy of these DNA damage inducing treatments could be enhanced by DNA damage repair inhibition. DT01 inhibits the complete DNA double-strand break repair machinery. Here, we assess the combination of DT01 with RFA, TACE or CT in preclinical models. Methods: For association with RFA, mice bearing flank-grafted tumors were sham treated (n=18), treated by DT01 (n=22), RFA (n=21) or a combination of DT01 and RFA (n=19). Mice were either sacrificed for pathological study or followed for survival. For association with TACE, rabbits bearing VX2 hepatocellular carcinoma in liver were untreated (n=9), treated with TACE (n=13) or treated with TACE and DT01 (n=14). Tumor growth, vascularization, necrosis and metastases were assessed with ultrasound scanning, color Doppler, pathology and autopsy respectively. For association with CT, mice bearing orthotopic liver tumors were administered NaCl (n=7), CT (doxorubicin) (n=10), systemic DT01 treatment (n=7) or an association of DT01 and CT (n=10). Tumor growth and pathological studies were assessed. Results: Mice treated by RFA and DT01 have longer survival compared to RFA alone (median survival: 57 vs 40 days) with 54% of complete responses while RFA alone improved survival moderately (median survival: 40 vs 28 days for control). Rabbits treated with TACE and DT01, in comparison to CT alone, show efficient tumor growth control, an increase of tumor necrosis (61% vs 40%), a decrease of metastases (21 vs 54%) and an absence of neoangiogenesis rebound. Mice treated with CT in combination with DT01 show a significant decrease in tumor volume and tumor necrosis, compared to the mice treated with CT alone. In all models, DT01 addition to treatments did not add any toxicity. Conclusions: Our results show that the addition of DT01 to RFA, TACE or CT enhances their antitumor activities and provide an experimental basis for the use of DT01 as an adjuvant therapy at each stage of HCC treatment.

The Mechanism of Radiosensitization by YM155, a Novel Small Molecule Inhibitor of Survivin Expression, is Associated with DNA Damage Repair

Background/Aims: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We investigated the effects of YM155, a small molecule inhibitor of survivin expression, on the radiosensitivity of human non-small cell lung cancer (NSCLC) cell lines and elucidated a relationship between the cellular localization of survivin and DNA double-strand break repair. Methods: The cellular distribution of survivin was determined by Western blotting of subcellular fractions and by immunofluorescent staining in A549 NSCLC cells. Radiation-induced DNA damage was evaluated based on histone H2AX phosphorylation and foci formation. The relationship between the cellular localization of survivin and DNA double-strand break repair was analyzed by Western blotting and co-immunoprecipitations. Results: YM155 down-regulated survivin expression in NSCLC cells in a concentration- and time-dependent manner. An in vitro clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to radiation. After irradiation, we observed a rapid accumulation of survivin in the nucleus. An immunofluorescent analysis of histone γ-H2AX demonstrated that the inhibition of survivin expression by YM155 resulted in impaired DNA double-strand break repair. Co-immunoprecipitation assays using nuclear extracts revealed an interaction between survivin, Ku70, γ-H2AX, and DNA-PKcs. Furthermore, S2056 autophosphorylation of DNA-PKcs was reduced in survivin-depleted cells. Conclusions: These results suggested that YM155 sensitized NSCLC cells to radiation, at least in part by inhibiting DNA repair and enhancing apoptosis via the down-regulation of survivin expression. YM155 pretreatment inhibited DNA-PKcs autophosphorylation at S2056. Nuclear survivin was involved in DNA double-strand break repair via interactions with members of the DNA double-strand break repair machinery.

PTH1–34 Blocks Radiation-induced Osteoblast Apoptosis by Enhancing DNA Repair through Canonical Wnt Pathway

Focal radiotherapy for cancer patients has detrimental effects on bones within the radiation field and the primary clinical signs of bone damage include the loss of functional osteoblasts. We reported previously that daily injection of parathyroid hormone (PTH, 1-34) alleviates radiation-induced osteopenia in a preclinical radiotherapy model by improving osteoblast survival. To elucidate the molecular mechanisms, we irradiated osteoblastic UMR 106-01 cells and calvarial organ culture and demonstrated an anti-apoptosis effect of PTH1-34 on these cultures. Inhibitor assay indicated that PTH exerts its radioprotective action mainly through protein kinase A/β-catenin pathway. γ-H2AX foci staining and comet assay revealed that PTH efficiently promotes the repair of DNA double strand breaks (DSBs) in irradiated osteoblasts via activating the β-catenin pathway. Interestingly, Wnt3a alone also blocked cell death and accelerated DNA repair in primary osteoprogenitors, osteoblastic and osteocytic cells after radiation through the canonical signaling. Further investigations revealed that both Wnt3a and PTH increase the amount of Ku70, a core protein for initiating the assembly of DSB repair machinery, in osteoblasts after radiation. Moreover, down-regulation of Ku70 by siRNA abrogated the prosurvival effect of PTH and Wnt3a on irradiated osteoblasts. In summary, our results identify a novel role of PTH and canonical Wnt signaling in regulating DSB repair machinery and apoptosis in osteoblasts and shed light on using PTH1-34 or Wnt agonist as possible therapy for radiation-induced osteoporosis.

MicroRNAs: The Short Link between Cancer and RT-Induced DNA Damage Response.

microRNAs: The Short Link between Cancer and RT-Induced DNA Damage Response.

A fine-scale dissection of the DNA double-strand break repair machinery and its implications for breast cancer therapy.

DNA-damage response machinery is crucial to maintain the genomic integrity of cells, by enabling effective repair of even highly lethal lesions such as DNA double-strand breaks (DSBs). Defects in specific genes acquired through mutations, copy-number alterations or epigenetic changes can alter the balance of these pathways, triggering cancerous potential in cells. Selective killing of cancer cells by sensitizing them to further DNA damage, especially by induction of DSBs, therefore requires careful modulation of DSB-repair pathways.Here, we review the latest knowledge on the two DSB-repair pathways, homologous recombination and non-homologous end joining in human, describing in detail the functions of their components and the key mechanisms contributing to the repair. Such an in-depth characterization of these pathways enables a more mechanistic understanding of how cells respond to therapies, and suggests molecules and processes that can be explored as potential therapeutic targets. One such avenue that has shown immense promise is via the exploitation of synthetic lethal relationships, for which the BRCA1–PARP1 relationship is particularly notable. Here, we describe how this relationship functions and the manner in which cancer cells acquire therapy resistance by restoring their DSB repair potential.

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage

DNA double-strand breaks (DSBs) representa serious threat for hematopoietic stem cells (HSCs). How cytokines and environmental signals integrate the DNA damage response and contribute to HSC-intrinsic DNA repair processes remains unknown. Thrombopoietin (TPO) and its receptor, Mpl, are critical factors supporting HSC self-renewal and expansion. Here, we uncover an unknown function for TPO-Mpl in the regulation of DNA damage response. We show that DNA repair following γ-irradiation (γ-IR) or the action of topoisomerase-II inhibitors is defective in Mpl(-/-) and in wild-type mouse or human hematopoietic stem and progenitor cells treated in the absence of TPO. TPO stimulates DNA repair invitro and invivo by increasing DNA-PK-dependent nonhomologous end-joining efficiency. This ensures HSC chromosomal integrity and limits their long-term injury in response to IR.This shows that niche factors can modulate theHSC DSB repair machinery and opens new avenues for administration of TPO agonists for minimizing radiotherapy-induced HSC injury and mutagenesis.

Histone H2A.Z Controls a Critical Chromatin Remodeling Step Required for DNA Double-Strand Break Repair

Chromatin remodeling during DNA double-strand break (DSB) repair is required to facilitate access to and repair of DSBs. This remodeling requires increased acetylation of histones and a shift in nucleosome organization to create open, relaxed chromatin domains. However, the underlying mechanism driving changes in nucleosome structure at DSBs is poorly defined. Here, we demonstrate that histone H2A.Z is exchanged onto nucleosomes at DSBs by the p400 remodeling ATPase. H2A.Z exchange at DSBs shifts the chromatin to an open conformation and is required for acetylation and ubiquitination of histones and for loading of the brca1 complex. H2A.Z exchange also restricts single-stranded DNA production by nucleases and is required for loading of the Ku70/Ku80 DSB repair protein. H2A.Z exchange therefore promotes specific patterns of histone modification and reorganization of the chromatin architecture, leading to the assembly of a chromatin template that is an efficient substrate for the DSB repair machinery.

Formation of Complex Extrachromosomal T-DNA Structures in Agrobacterium tumefaciens-Infected Plants

Agrobacterium tumefaciens is a unique plant pathogenic bacterium renowned for its ability to transform plants. The integration of transferred DNA (T-DNA) and the formation of complex insertions in the genome of transgenic plants during A. tumefaciens-mediated transformation are still poorly understood. Here, we show that complex extrachromosomal T-DNA structures form in A. tumefaciens-infected plants immediately after infection. Furthermore, these extrachromosomal complex DNA molecules can circularize in planta. We recovered circular T-DNA molecules (T-circles) using a novel plasmid-rescue method. Sequencing analysis of the T-circles revealed patterns similar to the insertion patterns commonly found in transgenic plants. The patterns include illegitimate DNA end joining, T-DNA truncations, T-DNA repeats, binary vector sequences, and other unknown "filler" sequences. Our data suggest that prior to T-DNA integration, a transferred single-stranded T-DNA is converted into a double-stranded form. We propose that termini of linear double-stranded T-DNAs are recognized and repaired by the plant's DNA double-strand break-repair machinery. This can lead to circularization, integration, or the formation of extrachromosomal complex T-DNA structures that subsequently may integrate.

Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements

DNA double strand breaks (DSB) can lead to development of genomic rearrangements, which are hallmarks of cancer. TMPRSS2-ERG gene fusions in prostate cancer (PCa) are among the most common genomic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor (AR) and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSB. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process requiring TOP2B and components of DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia (PIN) cells showed strong co-expression of AR and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSB in generating TMPRSS2-ERG rearrangements.

Radiation-Induced Survivin Nuclear Accumulation is Linked to DNA Damage Repair

Increased expression of survivin has been identified as a negative prognostic marker in a variety of human cancers. We have previously shown that survivin is a radiation-resistance factor and that the therapeutic effect of survivin knock-down might result from an impaired DNA repair capacity. In this study, we aimed to elucidate an interrelationship between survivin's cellular localization and DNA double-strand break repair. Survivin's cellular distribution and nuclear complex formation were assayed by Western blotting of subcellular fractions, by immunofluorescence staining, and co-immunoprecipitation in SW480 colorectal cancer cells. DNA repair capacity was analyzed by kinetics of gamma-H2AX foci formation, and by DNA-dependent protein kinase (DNA-PKcs) assays in the presence of survivin-specific or nonspecific control siRNA. Following irradiation, we observed a rapid nuclear accumulation of survivin and subsequent phosphorylation of the protein in the nucleus. Co-immunoprecipitation analyses from nuclear extracts revealed an interaction among survivin, Ku70, gamma-H2AX, MDC1, and DNA-PKcs that was confirmed by immunofluorescence co-localization in nuclear foci. Survivin knock down by siRNA resulted in an impaired DNA double strand break repair, as demonstrated by an increased detection of gamma-H2AX foci/nucleus at 60 min and a higher amount of residual gamma-H2AX foci at 24 hr postirradiation. Furthermore, we detected in survivin-depleted cells a hampered S2056 autophosphorylation of DNA-PKcs and a significantly decreased DNA-PKcs kinase activity. These data indicate that nuclear survivin is linked to DNA double-strand break repair by interaction with members of the DNA double-strand breaks repair machinery, thus regulating DNA-PKcs activity.