Nonamer dependent RAG cleavage at CpGs can explain mechanism of chromosomal translocations associated to lymphoid cancers.

Chromosomal translocations are considered as one of the major causes of lymphoid cancers. RAG complex, which is responsible for V(D)J recombination, can also cleave non-B DNA structures and cryptic RSSs in the genome leading to chromosomal translocations. The mechanism and factors regulating the illegitimate function of RAGs resulting in oncogenesis are largely unknown. Upon in silico analysis of 3760 chromosomal translocations from lymphoid cancer patients, we find that 93% of the translocation breakpoints possess adjacent cryptic nonamers (RAG binding sequences), of which 77% had CpGs in proximity. As a proof of principle, we show that RAGs can efficiently bind to cryptic nonamers present at multiple fragile regions and cleave at adjacent mismatches generated to mimic the deamination of CpGs. ChIP studies reveal that RAGs can indeed recognize these fragile sites on a chromatin context inside the cell. Finally, we show that AID, the cytidine deaminase, plays a significant role during the generation of mismatches at CpGs and reconstitute the process of RAG-dependent generation of DNA breaks both in vitro and inside the cells. Thus, we propose a novel mechanism for generation of chromosomal translocation, where RAGs bind to the cryptic nonamer sequences and direct cleavage at adjacent mismatch generated due to deamination of meCpGs or cytosines.

MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development.

Recombination activating genes (RAGs), consisting of RAG1 and RAG2, are stringently regulated lymphoid-specific genes, which initiate V(D)J recombination in developing lymphocytes. We report the regulation of RAG1 through a microRNA (miRNA), miR-29c, in a B cell stage-specific manner in mice and humans. Various lines of experimentation, including CRISPR-Cas9 genome editing, demonstrate the target specificity and direct interaction of miR-29c to RAG1. Modulation of miR-29c levels leads to change in V(D)J recombination efficiency in pre-B cells. The miR-29c expression is inversely proportional to RAG1 in a B cell developmental stage-specific manner, and miR-29c null mice exhibit a reduction in mature B cells. A negative correlation of miR-29c and RAG1 levels is also observed in leukemia patients, suggesting the potential use of miR-29c as a biomarker and a therapeutic target. Thus, our results reveal the role of miRNA in the regulation of RAG1 and its relevance in cancer.

Characterization of G-quadruplex antibody reveals differential specificity for G4 DNA forms.

Accumulating evidence suggests that human genome can fold into non-B DNA structures, when appropriate sequence and favourable conditions are present. Among these, G-quadruplexes (G4-DNA) are associated with gene regulation, chromosome fragility and telomere maintenance. Although several techniques are used in detecting such structures in vitro, understanding their intracellular existence has been challenging. Recently, an antibody, BG4, was described to study G4 structures within cells. Here, we characterize BG4 for its affinity towards G4-DNA, using several biochemical and biophysical tools. BG4 bound to G-rich DNA derived from multiple genes that form G-quadruplexes, unlike complementary C-rich or random sequences. BLI studies revealed robust binding affinity (Kd = 17.4 nM). Gel shift assays show BG4 binds to inter- and intramolecular G4-DNA, when it is in parallel orientation. Mere presence of G4-motif in duplex DNA is insufficient for antibody recognition. Importantly, BG4 can bind to G4-DNA within telomere sequence in a supercoiled plasmid. Finally, we show that BG4 binds to form efficient foci in four cell lines, irrespective of their lineage, demonstrating presence of G4-DNA in genome. Importantly, number of BG4 foci within the cells can be modulated, upon knockdown of G4-resolvase, WRN. Thus, we establish specificity of BG4 towards G4-DNA and discuss its potential applications.

Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs.

Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.

Biochemical activity of RAGs is impeded by Dolutegravir, an HIV integrase inhibitor.

HIV is a retrovirus that infects CD4+ T lymphocytes in human beings and causes immunodeficiency. In the recent years, various therapies have been developed against HIV, including targeting the HIV specific protein, integrase, responsible for integration of HIV cDNA into host DNA. Although, integrase is specific to HIV, it has functional and structural similarity with RAG1, one of the partner proteins associated with V(D)J recombination, a process by which immune diversity is generated in humans. Currently, there are three HIV integrase inhibitors: Elvitegravir, Dolutegravir, and Raltegravir, in the market which have been approved by the FDA (USA). All three drugs are used in anti-retroviral therapy (ART). Previously, we showed that amongst the HIV inhibitors, Elvitegravir could significantly decrease B cell maturation in vivo and inhibit the physiological activities of RAGs in vitro, unlike Raltegravir. In the present study, we address the effect of second-generation integrase inhibitor, Dolutegravir on RAG activities. Binding and nicking studies showed that, Dolutegravir could decrease the binding efficiency of RAG1 domains and cleavage on DNA substrates, but not as considerably as Elvitegravir. Thus, we show that although the integrase inhibitors such as Elvitegravir show an affinity towards RAG1, the newer molecules may have lesser side-effects.