Canonical and noncanonical forms of G4 DNA at cluster III of the BCL6 breakpoint region could lead to chromosomal translocation in DLBCL.

Cancer arises from the accumulation of genetic alterations, including chromosomal translocations and deletions. Faulty repair of DNA double-strand breaks can give rise to such chromosomal rearrangements. In this study, we focus on diverse translocations that share a common partner, BCL6 on chromosome 3, which are implicated in diffuse large B-cell lymphoma (DLBCL). Analysis of patient breakpoints identified several breakpoint clusters within BCL6, of which Cluster III is the focus of this work. Here, we investigate the role of non-B DNA structures in imparting chromosomal fragility. In silico analyses, gel shift assays, and circular dichroism confirmed G-quadruplex (G4) formation at BCL6 Cluster III. Mutation studies revealed multiple G4 conformations utilizing distinct G-stretches, including GNG motifs. Disrupting G4-forming sequences in this region enhanced plasmid propagation in E. coli, indicating structure-dependent replication stalling. Sodium bisulfite modification assays detected single-stranded character here, both in plasmids and chromosomal DNA, suggesting additional fragility hotspots within Cluster III. Ex vivo assays showed that the G4 structure blocks transcription as a roadblock. Together, these data demonstrate that G4 folding in BCL6 Cluster III generates partially single-stranded regions, rendering the locus prone to breakage and translocation.