Investigating DNA structural basis for fragility during chromosomal translocations associated with human B cell lymphomas

Abstract

Genome integrity is essential for normal cellular functions. Erroneous repair of DNA double-strand breaks (DSBs) could lead to chromosomal rearrangements, including chromosomal translocations that may result in altered protein expression or chimeric proteins. Cell-type specific translocations, as observed in human lymphomas, are important for oncogenic progression. One such instance is of t(14;18) translocation involving BCL2 (chromosome 18) and IgH (chromosome 14) loci, that causes follicular lymphoma (FL). In another illustrious example, diverse translocations involving a common partner, BCL6 (chromosome 3), have been reported in diffuse large B cell lymphoma (DLBL). In the present study, impact of deviations in DNA structure in facilitating chromosomal fragility was investigated. In silico, in vitro and ex vivo approaches were employed to study the translocation breakpoint regions. Ex vivo assays were conducted to evaluate the effect of non-B DNA structures on physiological process such as transcription. Patient breakpoint analyses for BCL2 and BCL6 genes revealed breakpoint clusters in both the translocation cases. Bioinformatic studies suggest formation of G-quadruplex at BCL6 cluster III, while the nature of structure was unclear in case of BCL2 MBR. Gel shift assays showed faster mobility in case of both the translocation breakpoint sequences under study when compared to corresponding control sequences. Circular dichroism studies showed a characteristic G-quadruplex spectrum for BCL6. However, for BCL2, an uncharacteristic spectrum with a positive plateau pattern was observed, which was previously reported in literature for cruciform-like structures. Sodium bisulfite modification assay revealed single-strandedness at the fragile region when plasmid with BCL6 breakpoint was probed. These sequences had not been previously predicted by bioinformatic analysis; subsequent study of these revealed GNG motifs that aided in G4-structure formation...