PIF1 is a conserved 5'-to-3' DNA helicase essential for maintaining nuclear and mitochondrial genome stability 1. PIF1 functions primarily through ATP-dependent unwinding of DNA and RNA-DNA hybrid structures, with particular efficiency against G-quadruplex (G4) DNA structures 2. By resolving G4 structures at guanine-rich genomic regions, PIF1 prevents replication fork stalling and double-strand breaks that would otherwise compromise genomic stability 3. Beyond G4 resolution, PIF1 participates in R-loop metabolism, resolving RNA-DNA hybrids formed during transcription and other cellular processes 4. PIF1 plays a critical regulatory role in telomere maintenance by negatively regulating telomerase activity—it unwinds the RNA-DNA hybrid intermediate of the telomerase reaction, thereby inhibiting telomere elongation and blocking telomere addition to double-strand breaks 1. Additional functions include facilitating Okazaki fragment processing and promoting replication fork progression through various barriers including tRNA genes and rDNA loci 5. PIF1 recruitment to DNA sites depends on interactions with replication protein A (RPA) and proliferating cell nuclear antigen (PCNA), enabling its dual-anchoring at replication forks 6. These multifaceted roles underscore PIF1's importance in coordinating DNA replication, repair, and telomere regulation for cellular viability.