Mechanistic Insights into Poly-(rC)-binding protein 1 driven Unfolding of Selected i-motif DNA at G1/S checkpoint

I-motifs are non-canonical, four-stranded DNA structures in cytosine-rich genomic regions, yet their protein-mediated regulation remains underexplored. Here, we identified PCBP1 as a selective i-motif-binding protein that unfolds specific i-motif structures depending on their protonation and hairpin-forming propensities. Systematic truncation analyses revealed that individual K-homology (KH) domains of PCBP1 cannot selectively bind or unfold i-motifs, but their coordinated actions restored wild-type PCBP1 functions. Using biochemical, biophysical, and molecular dynamics studies, we demonstrated that KH1 + 2 domains remodel i-motifs, recruiting KH3 to facilitate unfolding and efficient DNA replication. Chromatin and cell-based investigations revealed that PCBP1-knockdown increases i-motif formation at specific genomic loci, coinciding with G ₁ /S arrest and elevated γH2AX, indicative of genomic instability. During G ₁ /S transition, PCBP1 occupancy peaked at these i-motif loci, ensuring i-motif resolution in early S phase. These findings establish PCBP1 as a critical regulator of i-motif dynamics, directly linking its unfolding activity to G ₁ /S transition and genome stability.