G-quadruplex DNA formation induced by transcription in vitro and in vivo: Implications for a DNA structure mediated mechanism of recombination

Recombination is a cellular mechanism by which information contained on DNA molecules is exchanged or transferred. Recombination occurs in most cell types and falls into three categories: homologous, site-specific, and non-homologous or “illegitimate” recombination. In addition, in B cells activated by antigen, a process of region-specific recombination occurs known as class switch recombination. Class switch recombination causes the cell to switch from production of one class of antibody to another. The targets for switch recombination are guanine-rich DNA sequences, called switch (S) regions.; Telomeres, like switch regions, consist of G-rich repeats. Telomeres are also targets for region-specific recombination. In cells lacking telomerase, telomere maintenance depends on a mechanism that employs DNA recombination.; Oligonucleotides derived from G-rich S region and telomeric sequences have been shown to form higher order structures in vitro, called G-quadruplex or G4 DNA. We have proposed that the region-specific recombination that occurs at switch and telomeric regions proceeds through a pathway in which structures formed by G-rich regions are targets for recombination enzymes.; Here I demonstrate that G-rich switch regions and telomeric sequences carried within a plasmid genome readily form G-quadruplex structures both in vitro and in vivo. Formation of such structures requires transient DNA denaturation, induced in my experiments by transcription, which recapitulates the transcriptional activation prerequisite to switch recombination in vivo. By electron microscopy, I have shown that characteristic R-loops form in transcribed G-rich regions, in which one strand contains an extensive RNA:DNA hybrid and the other G-quadruplex DNA. The readiness with which such structures form in vivo argues for their involvement in recombination, and rationalizes the dependence of class switch recombination on transcription.; Direct identification of G-quadruplex DNA in vivo resolves the question of whether the sequence composition of G-rich genomic regions could confer distinctive biological properties. It also raises the possibility that if G-quadruplex DNA is a target for recombination in the cell, structure formation within G-rich DNA regions could contribute to genomic instability leading to genetic disease and malignancy.