Putative Cellular Roles of G-Quadruplexes

Genome-wide analyses have detected potential G-quadruplex forming sequences in eukaryotic and prokaryotic genomes of highly divergent organisms (24,45,119,120). The fact that their formation is cation dependent and stabilization especially occurs by K⁺ and Na⁺ favors their formation under physiological conditions. Formation of intramolecular G-quadruplexes requires the nucleic acid to be single-stranded, thus sites that are prone to G-quadruplex formation are promoter regions, the replication fork and recombination sites where duplex DNA becomes unzipped, mRNA which is naturally single-stranded and in addition the single-stranded overhangs of telomers in eukaryotes (121). Indeed in eukaryotes, especially in the human genome, quadruplexes have been found in functional genomic domains, such as oncogene promoter regions or at the telomers, which in the past has turned the spotlight on using them as druggable sites e.g. for cancer therapeutics (43,79,122,123). Proof for abundant quadruplex formation in vivo is increasing. For example in 2011 Rodriguez et al. found that cells treated with the small molecule G-quadruplex binder pyridostatin showed transcription- and replication dependent DNA damage.

Chromatin immunoprecipitation of the DNA damage marker γH2AX followed by high throughput sequencing analyses showed that the DNA associated to γH2AX was enriched for putative G-quadruplex binding sequences. Furthermore cell imaging with labelled pyridostatin showed co-localization with the helicase hPif1 (124). Pif1 from yeast is known to resolve G-quadruplexes during replication (67). In 2013 Balasubramanian and co-workers were the first to show the distribution of G-quadruplexes on eukaryotic chromosomes and their regulation during cell cycle progression in different types of human cell lines using a G-quadruplex-specific antibody (64). In a follow-up study the group also demonstrates the visualization of RNA G-quadruplex structures within the cytoplasm of human cells (65). Putative cellular roles that have been assigned to quadruplex structures are the involvement in organization and protection of the telomeres, stalling of the replication fork machinery, promotion of homologous recombination and regulation of transcription (25). Figure 8 gives an overview of the proposed cellular functions of quadruplexes. Possible scenarios will be explained in the following, then examples of reported G-quadruplex functions from the literature will be presented. As the circular genomes of bacteria

Figure 8: Putative Functions of G-Quadruplexes

G-rich sequences in non-telomeric regions of the genome might transiently form G-quadruplex structures (blue) after DNA duplex (gray and black) separation during replication, recombination or transcription. In addition mRNA (violet) is prone to G-quadruplex formation during translation. A: During replication G-quadruplexes may form in the

(green) progression or lead to gapped replication, if the template strand is looped out and replication is reinitiated at a downstream position. B: Recombination can be initiated by G-quadruplexes either by the secondary structure itself serving as homologous region, which will induce strand exchanges between different genomic regions (gray and black), or by double strand breaks (red lightning) that occur in proximity to the G-quadruplex, which will recruit the DNA repair machinery and lead to illegitimate recombination. Recombination may also be facilitated by keeping the DNA duplex in the single stranded conformation allowing for strand exchange. Adapted from (25). C: In promoter regions G-quadruplexes may repress transcription directly by forming an obstacle for the RNA polymerase or indirectly by recruiting protein factors that will inhibit the polymerase. Stimulation of transcription can be achieved by keeping the DNA duplex in the single stranded conformation or again indirectly by recruiting activating factors. Adapted from (25).

D: On the mRNA G-quadruplexes can inhibit translation either by blocking access of the ribosome (orange) to the RBS (orange line) or forming an obstacle for ribosome progression downstream of the RBS, which will lead to a truncated protein (gray circles) and possibly RNA degradation by RNases (gray pacman). The nascent polypeptide chain is represented by colored circles. Translation initiation could be achieved by freeing an RBS that is trapped in another secondary structure. Stalling of the ribosome during translation can induce frameshifts and lead to an altered gene product.

On the DNA level a G-quadruplex may interfere with replication, recombination or transcription.

Inhibition of replication can be achieved by a G-quadruplex structure formed in the strand that serves as template for the leading strand by stalling of the DNA polymerase (Figure 8A left). In the lagging strand replication takes place discontinuously. Here, G-quadruplex formation of the template strand may lead to gapped replication. Part of the template strand may loop out into a non-canonical structure and be bypassed by the polymerase, replication would be reinitiated at the next Okazaki fragment (Figure 8A right).

G-quadruplex formation has also been implicated during recombination: Due to their repetitive nature G-quadruplexes may represent regions of sequence overlap with other genomic regions.

Recombination could be initiated at the homologous secondary structures and lead to sequence exchange between different genomic regions associated to G-quadruplexes. A G-quadruplex may also facilitate recombination by keeping the duplex in the single stranded conformation and thereby promote strand exchange reactions. Furthermore strand breaks or deletions have been associated to alternative secondary structures. The DNA repair machinery may be recruited to the non-canonical structure and illegitimate recombination can occur (Figure 8B).

When located within promoter regions G-quadruplexes can function as transcriptional regulators.

Similarly to the situation during replication G-quadruplex formation in the template strand may again directly inhibit RNA polymerase progression. However, if the G-quadruplex were to be located on the non-template strand, transcription would be facilitated as the non-canonical structure would keep the DNA strands from realigning. Transcription could also be stimulated or repressed indirectly by binding of the G-quadruplex by protein factors, which in turn either recruit or inhibit the RNA polymerase (Figure 8C).

Finally, quadruplexes may also be located on the mRNA and then influence translation. G-quadruplex forming sequences positioned adjacent to or within the ribosomal binding site (RBS), may block the ribosome from binding to the mRNA. In this case translation initiation would be blocked. Conversely, the RBS may also be trapped in a secondary structure, such as a stem-loop, and not be accessible for the ribosome. In this case G-quadruplex formation in proximity to the

formation of a non-canonical structure within the mRNA located downstream of the ribosome may cause stalling of the ribosome. Translation may be stopped completely, leading to production of a truncated protein and potential degradation of the blocked mRNA. Ribosomal stalling may also induce frameshifting resulting in an altered gene product upon continuation of translation (Figure 8D bottom).