The functional significance of the RPA- and PCNA-dependent recruitment of Pif1 to DNA

Pif1 family helicases are multifunctional proteins conserved in eukaryotes, from yeast to humans. They are important for the genome maintenance in both nuclei and mitochondria, where they have been implicated in Okazaki fragment processing, replication fork progression and termination, telomerase regulation and DNA repair. While the Pif1 helicase activity is readily detectable on naked nucleic acids in vitro, the in vivo functions rely on recruitment to DNA. We identify the single-stranded DNA binding protein complex RPA as the major recruiter of Pif1 in budding yeast, in addition to the previously reported Pif1-PCNA interaction. The two modes of the Pif1 recruitment act independently during telomerase inhibition, as the mutations in the Pif1 motifs disrupting either of the recruitment pathways act additively. In contrast, both recruitment mechanisms are essential for the replication-related roles of Pif1 at conventional forks and during the repair by break-induced replication. We propose a molecular model where RPA and PCNA provide a double anchoring of Pif1 at replication forks, which is essential for the Pif1 functions related to the fork movement. Pif1 helicase is recruited to DNA by the ssDNA binding protein RPA and the replication clamp PCNA. This study reveals the functional roles of RPA and PCNA dependent recruitment of Pif1 to DNA.RPA is the major recruiter of the Pif1 helicase to DNA, while PCNA plays a lesser role. RPA and PCNA act in two independent pathways to recruit Pif1 to DNA breaks and telomeres. Both RPA and PCNA are essential for the replication-dependent roles of Pif1 at the hard-to-replicate regions and during DNA repair by break-induced replication. At replication forks, RPA and PCNA might work together anchoring Pif1 on DNA. Pif1 helicase is recruited to DNA by the ssDNA binding protein RPA and the replication clamp PCNA. This study reveals the functional roles of RPA and PCNA dependent recruitment of Pif1 to DNA.