The Structural Recognition of G‐quadruplex DNA by PC4: How Does a ssDNA Binding Protein Decide?

Over the past two decades, guanine‐rich nucleic acids have received considerable attention due to their potential to fold into a secondary DNA structure that has been termed a G‐quadruplex. G‐quadruplex sequences are distributed throughout the genome in distinct, non‐random locations such as the promoters of proto‐oncogenes and telomeric regions of chromosomes. Consequently, because of their non‐random location within the genome G‐quadruplex structures have been implicated in the regulation of genomic processes and their mis‐regulation can result in detrimental disease phenotypes such as cancer or neurodegenerative disorders. G‐quadruplexes are primarily formed by appropriately spaced di‐ or tri‐ guanine nucleotide repeats that self‐associate through Hoogsteen hydrogen bonding to form a planar G‐quartet. G‐quartets then stack onto one another, aided by the stabilization of monovalent cations, such as K + or Na + to form a complete G‐quadruplex. Utilizing the G‐quadruplex forming sequence from the c‐MYC gene promoter, we identified p ositive c oactivator of transcription (PC4), a single‐stranded DNA (ssDNA) binding protein, as a novel G‐quadruplex interactor that binds with a low nano‐molar K d value of ~2 nM, similar to that observed with ssDNA. Based on the structural differences between G‐quadruplex and ssDNA, we surmised that substantial differences might exist between the binding mode for each DNA form. Thus, we set out to investigate what differences exist between these two complexes. Equilibrium binding experiments with alternative G‐quadruplex structures did not influence the K d value for the binding interaction. Fluorescence resonance energy transfer (FRET) stopped‐flow unwinding experiments revealed that formation of the PC4‐G‐quadruplex complex does not result in complete destabilization of the G‐quadruplex structure. Intrinsic protein fluorescence quenching experiments revealed the stoichiometry of the PC4‐G‐quadruplex complex to be a 1:1 ratio. Mutation of key amino acid residues involved in DNA binding revealed a difference in the requirement for the base‐stacking residues W89 and F77 in ssDNA and G‐quadruplex DNA binding. Taken together, these results provide a novel mode of binding by PC4 through the recognition of DNA secondary structure. The binding data indicates that PC4 can interact with G‐quadruplex DNA in a manner distinct from its binding to ssDNA, consistent with a potential role for this activity in vivo . Support or Funding Information This work was supported by NIH Grant R01 GM098922 and the Department of Biochemistry and Molecular Biology, UAMS.