G-rich DNA repeats that can form G-quadruplex structures are prevalent in bacterial genomes and are frequently associated with regulatory regions of genes involved in virulence, antigenic variation, and antibiotic resistance. These sequences are also inherently mutagenic and can lead to changes affecting cell survival and adaptation. Transcription of the G-quadruplex-forming repeat (G₃T)_n in E. coli, when mRNA comprised the G-rich strand, promotes G-quadruplex formation in DNA and increases rates of deletion of G-quadruplex-forming sequences. The genomic instability of G-quadruplex repeats may be a source of genetic variability that can influence alterations and evolution of bacteria. The DNA chaperone Hfq is involved in the genetic instability of these G-quadruplex sequences. Inactivation of the hfq gene decreases the genetic instability of G-quadruplex, demonstrating that the genomic instability of this regulatory element can be influenced by the E. coli highly pleiotropic Hfq protein, which is involved in small noncoding RNA regulation pathways, and DNA organization and packaging. We have shown previously that the protein binds to and stabilizes these sequences, increasing rates of their genomic instability. Here, we extend this analysis to characterize the role of the C-terminal domain of Hfq protein in interaction with G-quadruplex structures. This allows to better understand the function of this specific region of the Hfq protein in genomic instability.

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G₃T)₄, (G₃T)₈, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.