Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA

Abstract

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA.

The Top1 paradox: Friend and foe of the eukaryotic genome

“…Alternatively, the observed effect could be due to the sequestering and reduced function of Top1-interacting proteins. As further confirmation that Top1 reduces the adverse effect of G4 DNA, loss of Top1 significantly increases sensitivity of yeast cells to the G4-stabilizing ligand TmPyP4 [53]. Defining how the G4 binding properties of Top1 affect genome instability is an important area of future study.…”

High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

“…For Chromatin IP (ChIP), a previously described protocol was used [34] with following modifications. Anti-FLAG antibody (clone M2 -Sigma) conjugated to Protein G Dynabeads (Life Technologies) were used in pull-down.…”

“…A defective BLM helicase failed to unwind G4-DNA and caused increasing recombination frequencies and a high incidence of cancer in Bloom's syndrome [33]. More recently, the co-transcriptional activator Sub1, which interacts with both G4 DNA and the G4-helicase Pif1, was shown to suppress the G4-associated genome instability by facilitating the recruitment of Pif1 helicase to co-transcriptionally formed G4 DNA structures [34].…”

Structure and function of Pif1 helicase

“…ScPif1 interacts physically with Rim1, the yeast mitochondrial ssDNA binding protein, and this interaction increases the product formation for dsDNA unwinding [28]. ScPif1 also interacts with Sub1 [109], a co-transcriptional activator that reduces genome instability at G4DNA sequences in yeast [109]. Sub1 binds tightly to G4DNA but does not unfold G4DNA structures [110].…”

“…This suggests that Sub1 may instead recruit a G4DNA resolving protein to the G4DNA site. Indeed, Sub1 and ScPif1 interact physically and genetic experiments confirmed that Sub1 and ScPif1 act in the same pathway to suppress G4DNA associated genome instability [109]. ScPif1 has also been shown to interact functionally with Polδ-PCNA through a physical interaction with PCNA [15].…”