The histone methyltransferase PRDM9 is a key determinant of meiotic recombination in humans by depositing activating histone marks that promote recruitment of the recombination machinery. PRDM9 recognizes DNA primarily through a repetitive zinc-finger array that binds specific sequence motifs. The complementary strands of some PRDM9 target motifs are G-rich and can adopt DNA secondary structures known as G-quadruplexes (G4s). These folded G4 structures have been hypothesized to provide alternative DNA recognition platforms and to influence the local chromatin environment relevant to recombination initiation.
This study investigated the relationship between PRDM9 binding sites and G4 motifs using computational analyses of predicted and experimentally validated G4s, together with biochemical binding assays, to test whether PRDM9 can directly recognize folded G4 structures and whether G4 stability influences binding.
The authors performed computational analyses that compared PRDM9 binding sites with databases of predicted and experimentally validated G4 motifs. These analyses revealed that G4 motifs are among the most prevalent features at PRDM9 binding sites. The strongest enrichments were observed for G4s classified as highly stable. Importantly, the enrichment of G4 motifs at PRDM9 sites was reported to be largely independent of G4 loop length, indicating that stability rather than loop architecture was a primary correlate of PRDM9-associated G4 prevalence.
The computational work supports a nonrandom association between PRDM9 target regions and G4-forming potential, suggesting that G4s could contribute to PRDM9 targeting or retention at recombination-associated loci.
To test direct binding, the investigators used electrophoretic mobility shift assays (EMSA) with short, single-stranded oligonucleotides capable of forming G4 structures, in addition to canonical double-stranded DNA targets. These biochemical assays demonstrated that PRDM9 directly bound folded G4 structures. The experiments indicate that PRDM9's DNA recognition capacity extends beyond classic double-stranded sequence-specific binding to include folded single-stranded G4 conformations.
The EMSA approach provides direct evidence of PRDM9–G4 interaction under in vitro conditions, corroborating the computational association of G4 motifs with PRDM9 sites.
Across different G4 motifs, PRDM9 binding strength correlated with measures of G4 stability. The authors observed increased PRDM9 binding when the same G4-forming sequence was stabilized either by increasing potassium concentration, which favors G4 folding, or by adding a known G4-stabilizing ligand. These manipulations produced greater binding signals in EMSA, supporting a mechanistic link between G4 stability and PRDM9 affinity.
This pattern was consistent across multiple G4 sequences tested, indicating a generalizable relationship between G4 thermodynamic or structural stability and PRDM9 recognition.
To further test structural dependence, the team examined PRDM9 binding to an artificial G4-forming sequence that does not occur in the human genome. PRDM9 bound this artificial G4, demonstrating that recognition does not require native genomic context. Conversely, mutating the artificial sequence to prevent G4 structure formation abolished PRDM9 binding, indicating that the folded G-quadruplex conformation—rather than the primary sequence per se—was necessary for interaction in these assays.
These results strengthen the conclusion that PRDM9 recognition can be structure-dependent and that preventing G4 folding removes the binding substrate.
Based on the combined computational and biochemical findings, the authors propose a model in which stable G4 structures facilitate PRDM9 recruitment to sites of future recombination. Stable G4s may create discrete regions of increased local chromatin accessibility or present an additional binding platform that complements zinc-finger sequence recognition. Together, these effects could contribute to the establishment of a chromatin environment permissive for the initiation of meiotic recombination.
The model links G4 structural biology with PRDM9-mediated chromatin marking and the downstream recruitment of the meiotic recombination machinery.
The study combined genome-wide computational motif analyses using predicted and experimentally validated G4 datasets with in vitro biochemical assays. Electrophoretic mobility shift assays were used to test direct binding of PRDM9 to folded G4 oligonucleotides, and the impact of G4 stabilizing conditions (potassium concentration, stabilizing ligand) and mutational disruption of G4 motifs were evaluated. The source summary does not provide additional technical parameters, quantitative binding affinities, or full experimental protocols.
The authors declared no competing interests. Funding sources included an Early Career Grant of the University of Salzburg, regional Cancer Cluster Salzburg support, a university priority program, an Erasmus+ Mobility Grant, and an award from Masaryk University noted in the source. The work is posted as a bioRxiv preprint (posted August 05, 2026) and is available under a CC-BY 4.0 International license.
This study provides computational and biochemical evidence that folded G-quadruplex DNA structures are prevalent at PRDM9 binding sites and that PRDM9 can directly bind G4s in a stability-dependent manner. Stabilization of G4 folding increases PRDM9 binding, and disruption of G4 formation abolishes binding to an artificial sequence, supporting a structural recognition mechanism. The authors propose that stable G4s contribute to PRDM9 recruitment and formation of a chromatin environment conducive to meiotic recombination. Specific quantitative details and full experimental methods were not reported in the source summary and are available in the full preprint.