Short loop length and high thermal stability determine genomic instability induced by G‐quadruplex‐forming minisatellites

Abstract

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo.

Recombination events among virulence genes in malaria parasites are associated with G-quadruplex-forming DNA motifs

“…To assess whether var -gene-associated PQSs might have more recombinogenic properties than PQSs located elsewhere, we scored both groups for the criteria that were reported in the yeast model system to define particularly recombinogenic motifs [30]: loop lengths of only 1 or 2 nucleotides, total loop length ≤7 nucleotides, single pyrimidine rather than purine loops, and having the longest of the three loops in the central position rather than a flanking position (Fig. 1).…”

“…Different G4 structures have different stabilities in vitro and in a yeast model system it was recently shown that this relates to their ability to promote recombination in vivo. Highly stable G4s, which generally featured very small loops containing single pyrimidines rather than single purines, were particularly prone to cause recombination events [30]. The authors then went on to establish that such sequences were under-represented in several other genomes from Caenorhabditis elegans to human, indicating that their particular recombinogenic properties may be selected against during genome evolution [30].…”

“…Firstly, are the PQSs in the P. falciparum genome likely to promote recombination, and might the ones associated with var genes be more recombinogenic than those elsewhere in the genome? We hypothesize that if genome-destabilizing PQSs are generally selected against, these sequences should be under-represented, as they are in other genomes [30]. However, if var -associated PQSs can play a specific positive role in promoting inter- var -gene shuffling, conferring a potential advantage in immune evasion, then counter-selection might occur for high recombinogenic potential amongst var -associated PQSs.…”

“…However, if var -associated PQSs can play a specific positive role in promoting inter- var -gene shuffling, conferring a potential advantage in immune evasion, then counter-selection might occur for high recombinogenic potential amongst var -associated PQSs. An analogous theory has been proposed to explain the presence of the unique short-loop G4 motif that is found upstream of the pilE locus in N. gonorrhoeae [30]. …”

The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism

“…Supporting this view, incorporation of sequences predicted to form particularly stable G4s leads to genetic instability in yeast, suggesting that these G4 structures are not processed efficiently and pose blocks to replication (12). Indeed, several helicases have been shown to possess G4 disruption activity (13).…”