Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells.

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Whole genome sequencing and analysis of plant growth promoting bacteria isolated from the rhizosphere of plantation crops coconut, cocoa and arecanut.

Coconut, cocoa and arecanut are commercial plantation crops that play a vital role in the Indian economy while sustaining the livelihood of more than 10 million Indians. According to 2012 Food and Agricultural organization's report, India is the third largest producer of coconut and it dominates the production of arecanut worldwide. In this study, three Plant Growth Promoting Rhizobacteria (PGPR) from coconut (CPCRI-1), cocoa (CPCRI-2) and arecanut (CPCRI-3) characterized for the PGP activities have been sequenced. The draft genome sizes were 4.7 Mb (56% GC), 5.9 Mb (63.6% GC) and 5.1 Mb (54.8% GB) for CPCRI-1, CPCRI-2, CPCRI-3, respectively. These genomes encoded 4056 (CPCRI-1), 4637 (CPCRI-2) and 4286 (CPCRI-3) protein-coding genes. Phylogenetic analysis revealed that both CPCRI-1 and CPCRI-3 belonged to Enterobacteriaceae family, while, CPCRI-2 was a Pseudomonadaceae family member. Functional annotation of the genes predicted that all three bacteria encoded genes needed for mineral phosphate solubilization, siderophores, acetoin, butanediol, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinase, phenazine, 4-hydroxybenzoate, trehalose and quorum sensing molecules supportive of the plant growth promoting traits observed in the course of their isolation and characterization. Additionally, in all the three CPCRI PGPRs, we identified genes involved in synthesis of hydrogen sulfide (H2S), which recently has been proposed to aid plant growth. The PGPRs also carried genes for central carbohydrate metabolism indicating that the bacteria can efficiently utilize the root exudates and other organic materials as energy source. Genes for production of peroxidases, catalases and superoxide dismutases that confer resistance to oxidative stresses in plants were identified. Besides these, genes for heat shock tolerance, cold shock tolerance and glycine-betaine production that enable bacteria to survive abiotic stress were also identified.

Transcriptome profiling reveals higher vertebrate orthologous of intra-cytoplasmic pattern recognition receptors in grey bamboo shark.

From an immunologist perspective, sharks are an important group of jawed cartilaginous fishes and survey of the public database revealed a great gap in availability of large-scale sequence data for the group of Chondrichthyans the elasmobranchs. In an attempt to bridge this deficit we generated the transcriptome from the spleen and kidney tissues (a total of 1,606,172 transcripts) of the shark, Chiloscyllium griseum using the Illumina HiSeq2000 platform. With a cut off of > = 300 bp and an expression value of >1RPKM we used 43,385 transcripts for BLASTX analysis which revealed 17,548 transcripts matching to the NCBI nr database with an E-value of < = 10(-5) and similarity score of 40%. The longest transcript was 16,974 bases with matched to HECT domain containing E3 ubiqutin protein ligase. MEGAN4 annotation pipeline revealed immune and signalling pathways including cell adhesion molecules, cytokine-cytokine receptor interaction, T-cell receptor signalling pathway and chemokine signaling pathway to be highly expressed in spleen, while different metabolism pathways such as amino acid metabolism, carbohydrate metabolism, lipid metabolism and xenobiotic biodegradation were highly expressed in kidney. Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction. We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway. The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

The homologous recombination protein RAD51D mediates the processing of 6-thioguanine lesions downstream of mismatch repair.

Thiopurines are extensively used as immunosuppressants and in the treatment of childhood cancers, even though there is concern about therapy-induced leukemias and myelodysplastic syndromes resulting from thiopurine use. Following metabolic activation, thiopurines are incorporated into DNA and invoke mismatch repair (MMR). Recognition of 6-thioguanine (6-thioG) in DNA by key MMR proteins results in cell death rather than repair. There are suggestions that homologous recombination (HR) is involved downstream of MMR following thiopurine treatment, but the precise role of HR is poorly understood. In this study, we demonstrate that cells deficient in RAD51D (a RAD51 paralogue) are extremely sensitive to 6-thioG. This sensitivity is almost completely rescued by the deletion of Mlh1, which suggests that HR is involved in the repair of the 6-thioG-induced recombinogenic lesions generated by MMR. Furthermore, 6-thioG induces chromosome aberrations in the Rad51d-deficient cells. Interestingly, Rad51d-deficient cells show a striking increase in the frequency of triradial and quadriradial chromosomes in response to 6-thioG therapy. The presence of these chromatid exchange-type aberrations indicates that the deficiency in RAD51D-dependent HR results in profound chromosomal damage precipitated by the processing of 6-thioG by MMR. The radials are notable as an important source of chromosomal translocations, which are the most common class of mutations found in hematologic malignancies. This study thus suggests that HR insufficiency could be a potential risk factor for the development of secondary cancers that result from long-term use of thiopurines in patients.

RAD51D protects against MLH1-dependent cytotoxic responses to O(6)-methylguanine.

S(N)1-type methylating agents generate O(6)-methyl guanine (O(6)-meG), which is a potently mutagenic, toxic, and recombinogenic DNA adduct. Recognition of O(6)-meG:T mismatches by mismatch repair (MMR) causes sister chromatid exchanges, which are representative of homologous recombination (HR) events. Although the MMR-dependent mutagenicity and toxicity caused by O(6)-meG has been studied, the mechanisms of recombination induced by O(6)-meG are poorly understood. To explore the HR and MMR genetic interactions in mammals, we used the Rad51d and Mlh1 mouse models. Ablation of Mlh1 did not appreciably influence the developmental phenotypes conferred by the absence of Rad51d. Mouse embryonic fibroblasts (MEFs) deficient in Rad51d can only proliferate in p53-deficient background. Therefore, Rad51d(-/-)Mlh1(-/-)Trp53(-/-) MEFs with a combined deficiency of HR and MMR were generated and comparisons between MLH1 and RAD51D status were made. To our knowledge, these MEFs are the first mammalian model system for combined HR and MMR defects. Rad51d-deficient MEFs were 5.3-fold sensitive to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) compared to the Rad51d-proficient MEFs. A pronounced G2/M arrest in Rad51d-deficient cells was accompanied by an accumulation of gamma-H2AX and apoptosis. Mlh1-deficient MEFs were resistant to MNNG and showed no G2/M arrest or apoptosis at the doses used. Importantly, loss of Mlh1 alleviated sensitivity of Rad51d-deficient cells to MNNG, in addition to reducing gamma-H2AX, G2/M arrest and apoptosis. Collectively, the data support the hypothesis that MMR-dependent sensitization of HR-deficient cells is specific for O(6)-meG and suggest that HR resolves DNA intermediates created by MMR recognition of O(6)-meG:T. This study provides insight into recombinogenic mechanisms of carcinogenesis and chemotherapy resulting from O(6)-meG adducts.

Cisplatin resistance conferred by the RAD51D (E233G) genetic variant is dependent upon p53 status in human breast carcinoma cell lines.

RAD51D, a paralog of the mammalian RAD51 gene, contributes towards maintaining genomic integrity by homologous recombination DNA repair and telomere maintenance. A RAD51D variant, E233G, was initially identified as a potential susceptibility allele in high-risk, site-specific, familial breast cancer. We describe in this report that the Rad51d (E233G) genetic variant confers increased cisplatin resistance and cell growth phenotypes in human breast carcinoma cell lines with a mutant p53 gene (BT20 and T47D) but not with a wild-type p53 gene (MCF-7). Treatment with a p53 specific inhibitor, pifithrin alpha, restored this resistant phenotype in the MCF-7 cell line. Additionally, Rad51d (E233G) conferred increased cisplatin resistance of an MCF7 cell line in which p53 expression was stably knocked down by shRNAp53, indicating that the effect of this variant is dependent upon p53 status. Further study of Rad51d (E233G) will provide mechanistic insight towards the role of RAD51D in cellular response to anticancer agents and as a potential target for cancer therapy.

The Alternative Splicing Mutation Database: a hub for investigations of alternative splicing using mutational evidence.

BACKGROUND: Some mutations in the internal regions of exons occur within splicing enhancers and silencers, influencing the pattern of alternative splicing in the corresponding genes. To understand how these sequence changes affect splicing, we created a database of these mutations. FINDINGS: The Alternative Splicing Mutation Database (ASMD) serves as a repository for all exonic mutations not associated with splicing junctions that measurably change the pattern of alternative splicing. In this initial published release (version 1.2), only human sequences are present, but the ASMD will grow to include other organisms, (see Availability and requirements section for the ASMD web address).This relational database allows users to investigate connections between mutations and features of the surrounding sequences, including flanking sequences, RNA secondary structures and strengths of splice junctions. Splicing effects of the mutations are quantified by the relative presence of alternative mRNA isoforms with and without a given mutation. This measure is further categorized by the accuracy of the experimental methods employed. The database currently contains 170 mutations in 66 exons, yet these numbers increase regularly.We developed an algorithm to derive a table of oligonucleotide Splicing Potential (SP) values from the ASMD dataset. We present the SP concept and tools in detail in our corresponding article. CONCLUSION: The current data set demonstrates that mutations affecting splicing are located throughout exons and might be enriched within local RNA secondary structures. Exons from the ASMD have below average splicing junction strength scores, but the difference is small and is judged not to be significant.

Calculation of splicing potential from the Alternative Splicing Mutation Database.

BACKGROUND: The Alternative Splicing Mutation Database (ASMD) presents a collection of all known mutations inside human exons which affect splicing enhancers and silencers and cause changes in the alternative splicing pattern of the corresponding genes. FINDINGS: An algorithm was developed to derive a Splicing Potential (SP) table from the ASMD information. This table characterizes the influence of each oligonucleotide on the splicing effectiveness of the exon containing it. If the SP value for an oligonucleotide is positive, it promotes exon retention, while negative SP values mean the sequence favors exon skipping. The merit of the SP approach is the ability to separate splicing signals from a wide range of sequence motifs enriched in exonic sequences that are attributed to protein-coding properties and/or translation efficiency. Due to its direct derivation from observed splice site selection, SP has an advantage over other computational approaches for predicting alternative splicing. CONCLUSION: We show that a vast majority of known exonic splicing enhancers have highly positive cumulative SP values, while known splicing silencers have core motifs with strongly negative cumulative SP values. Our approach allows for computation of the cumulative SP value of any sequence segment and, thus, gives researchers the ability to measure the possible contribution of any sequence to the pattern of splicing.

Regulatory circuit based on autogenous activation-repression: roles of C-boxes and spacer sequences in control of the PvuII restriction-modification system.

Type II restriction-modification (R-M) systems comprise a restriction endonuclease (REase) and a protective methyltransferase (MTase). After R-M genes enter a new cell, MTase must appear before REase or the chromosome will be cleaved. PvuII and some other R-M systems achieve this delay by cotranscribing the REase gene with the gene for an autogenous transcription activator (the controlling or 'C' protein C.PvuII). This study reveals, through in vivo titration, that C.PvuII is not only an activator but also a repressor for its own gene. In other systems, this type of circuit can result in oscillatory behavior. Despite the use of identical, symmetrical C protein-binding sequences (C-boxes) in the left and right operators, C.PvuII showed higher in vitro affinity for O(L) than for O(R), implicating the spacer sequences in this difference. Mutational analysis associated the repression with O(R), which overlaps the promoter -35 hexamer but is otherwise dispensable for activation. A nonrepressing mutant exhibited poor establishment in new cells. Comparing promoter-operator regions from PvuII and 29 R-M systems controlled by C proteins revealed that the most-highly conserved sequence is the tetranucleotide spacer separating O(L) from O(R). Any changes in that spacer reduced the stability of C.PvuII-operator complexes and abolished activation.

Novel testis/sperm-specific contraceptive targets identified using gene knockout studies.

The population explosion and unintended pregnancies resulting in elective abortions continue to be the major public health issues in spite of availability of current methods of contraception. There is an urgent need for a better method of contraception that is accepted, effective, and available. Various targets are being investigated that can be used for contraception. The gene knockout technology is a powerful approach to identify such novel targets. Using search in the database, at least 93 genes were identified in the literature whose deletion demonstrated an effect on fertility in male mice. However, majority of these knockouts also demonstrated an effect on non-reproductive organs concomitant with an effect on fertility. The knockouts of only a few genes/proteins induced a specific effect on fertility without a serious side effect. The potential role of these novel genes/proteins in contraception/contraceptive vaccine development is discussed.

Role of tyrosine phosphorylation in sperm capacitation / acrosome reaction.

Capacitation is an important physiological pre-requisite before the sperm cell can acrosome react and fertilize the oocyte. Recent reports from several laboratories have amply documented that the protein phosphorylation especially at tyrosine residues is one of the most important events that occur during capacitation. In this article, we have reviewed the data from our and other laboratories, and have constructed a heuristic model for the mechanisms and molecules involved in capacitation/acrosome reaction.