Nonhomologous DNA End Joining in Cell-Free Extracts

Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems

How does DNA break during chromosomal translocations?

Chromosomal translocations are one of the most common types of genetic rearrangements and are molecular signatures for many types of cancers. They are considered as primary causes for cancers, especially lymphoma and leukemia. Although many translocations have been reported in the last four decades, the mechanism by which chromosomes break during a translocation remains largely unknown. In this review, we summarize recent advances made in understanding the molecular mechanism of chromosomal translocations

Molecular mechanism of Endosulfan action in mammals

Endosulfan is a broad-spectrum organochlorine pesticide, speculated to be detrimental to human health in areas of active exposure. However, the molecular insights to its mechanism of action remain poorly understood. In two recent studies, our group investigated the physiological and molecular aspects of endosulfan action using in vitro, ex vivo and in vivo analyses. The results showed that apart from reducing fertility levels in male animals, Endosulfan induced DNA damage that triggers compromised DNA damage response leading to undesirable processing of broken DNA ends. In this review, pesticide use especially of Endosulfan in the Indian scenario is summarized and the importance of our findings, especially the rationalized use of pesticides in the future, is emphasized

Biochemical characterization of nonamer binding domain of RAG1 reveals its thymine preference with respect to length and position

RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA

Detection of G-quadruplex DNA using primer extension as a tool

DNA sequence and structure play a key role in imparting fragility to different regions of the genome. Recent studies have shown that non-B DNA structures play a key role in causing genomic instability, apart from their physiological roles at telomeres and promoters. Structures such as G-quadruplexes, cruciforms, and triplexes have been implicated in making DNA susceptible to breakage, resulting in genomic rearrangements. Hence, techniques that aid in the easy identification of such non-B DNA motifs will prove to be very useful in determining factors responsible for genomic instability. In this study, we provide evidence for the use of primer extension as a sensitive and specific tool to detect such altered DNA structures. We have used the G-quadruplex motif, recently characterized at the BCL2 major breakpoint region as a proof of principle to demonstrate the advantages of the technique. Our results show that pause sites corresponding to the non-B DNA are specific, since they are absent when the G-quadruplex motif is mutated and their positions change in tandem with that of the primers. The efficiency of primer extension pause sites varied according to the concentration of monovalant cations tested, which support G-quadruplex formation. Overall, our results demonstrate that primer extension is a strong in vitro tool to detect non-B DNA structures such as G-quadruplex on a plasmid DNA, which can be further adapted to identify non-B DNA structures, even at the genomic level

Synthesis and antiproliferative activity of novel homopiperazine derivatives in leukemia cells

A series of novel homopiperazine derivatives were synthesized and characterized using 1H NMR, LC MS, IR and elemental analysis data. These novel molecules were evaluated for their antiproliferative activity against Reh, leukemia cells using trypan blue and MTT assays. All the molecules showed cytotoxicity with IC50 values between 50-100 μM as calculated by trypan blue assay and greater than 100 μM as calculated by MTT assay. Compound 6b with 3,5-dinitro substituents on phenyl ring of the aryl carboxamide moiety attached to homopiperazine ring showed good activity with IC50 value of 41 μM

Novel ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate derivatives: synthesis and anticancer activities

To explore the anticancer activity of 2, 4, 5, 6-substituted pyrimidines, several ethyl 2-(1-aminocyclobutyl)-5-(benzoyloxy)-6-hydroxy-pyrimidine-4-carboxylate derivatives associated with the different substituted aromatic/aliphatic carboxamides and sulfonamides were synthesized. Different groups and position on phenyl ring attached to the carboxamide and sulfonamide of the pyrimidine led to two set of compounds. Their chemical structures were confirmed by IR,1H NMR and LC/MS analysis. Cytotoxicity of all the synthesized compounds were examined on human leukemia celllines (K562 and CEM). The preliminary results showed most of the derivatives exhibited good antitumor activity. Compound with para chloro substitution among carboxamides and compound with meta dichloro substitution among sulphonamidesexhibited significant antitumor activity with IC50 value of 14.0 μM and 15.0 μM respectively against K562cell line. For comparison among electron donating groups between carboxamides and sulfonamides, compounds with para tert-butyl substitution were chosen for further studies. Cell cycle analysis suggests that both tert-butyl substituted compounds are able to induce apoptosis

Plant-derived tetranortriterpenoid, methyl angolensate activates apoptosis and prevents ehrlich ascites carcinoma induced tumorigenesis in mice

Background: Cancer is a leading health problem throughout the world. For decades, natural plant products have been playing promising roles as anticancer agents. Objective: The present study aims to investigate the chemotherapeutic potential of Methyl Angolensate (MA), purified from Soymida febrifuga in mice bearing carcinoma and examines the molecular basis for its anticancer actions. Study Design: The inhibitory effects of MA treatment on the survival of mice bearing Carcinoma and adverse side effects of MA treatment in mice were analyzed. Methods: Tumor volume, life span, histopathology, Immunohistochemical (IHC) analysis, estimation of liver enzyme, alkaline phosphatase and metabolites, creatinine and urea. Results: Oral administration of MA in mice with Ehrlich Ascites Carcinoma showed significant inhibition of tumor growth compared to untreated mice. We observed a significant increase in the life span (∼4-fold) of tumor bearing animals following treatment with MA. MA affected tumor cell proliferation by activating intrinsic pathway of apoptosis without imparting any side effect on normal cells. MA treatment in mice showed no major side effects. Conclusion: MA treatment showed significant inhibition of tumor growth by inducing apoptosis as well increased life span of mice, with no adverse side effects to normal cells. Altogether, the present in vivo study provides new insights of MA serving as a cancer chemotherapeutic agent

Delineating the Mechanism of Fragility at BCL6 Breakpoint Region Associated With Translocations in Diffuse Large B Cell Lymphoma

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5\u27 UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt\u27s lymphoma

t(8;14) translocation is the hallmark of Burkitt\u27s lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt\u27s lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt\u27s lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation