Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication

Abstract

SUMMARY The S. cerevisiae Pif1 helicase functions with DNA polymerase (Pol) δ in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction. This mutation impairs the ability of Pif1 to enhance DNA strand displacement synthesis by Pol δ in vitro and also the efficiency of BIR in cells. These results provide insights into the role of the Pif1-PCNA-Pol δ ensemble during DNA break repair by homologous recombination.

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

“…PIF1 helicases share the conserved ATPase/helicase domain and an intrinsically disordered N-terminal tail of variable sequence. Post-translational modification sites, proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box and alternative start sites, which give rise to mitochondrial isoforms, are marked [9][10][11][12][13][14].…”

“…During BIR, ScPif1 is found at and downstream of the site of strand invasion, which led to the suggestion that ScPif1 is directly involved in D-loop migration [52,188]. Binding of ScPif1 to PCNA, which stimulates strand displacement DNA synthesis by DNA polymerase δ, also stimulates BIR [11]. Furthermore, ScPif1's processivity is regulated by force on ssDNA [189].…”

“…Binding sites on Rrm3, ScPif1, and Pfh1 and the method of detection are indicated. Putative binding proteins shown in darker colored boxes were identified in various high-throughput affinity capture experiments[9,11,19,40,42,52,93,.…”

TheDrosophila melanogasterPIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair

“…One speculative model, consistent with all these data, is that POL32 acts in the context of the Pol δ complex to promote processivity, while PIF1 is important for the progression of the mobile Dloop. PIF1 could act in concert with PCNA to promote Pol -mediated strand displacement at the front of the replication bubble (BUZOVETSKY et al 2017), and/or behind the D-loop to unwind the newly-synthesized strand to relieve topological hindrance of the nascent strand with the template strand (WILSON et al 2013). If POL32 is present, then Pol  can engage in strand displacement synthesis and the requirement for PIF1 activity is removed.…”

“…Recently, ScPif1 was shown to be required during break-induced replication (BIR), a type of HR repair observed at one-ended DNA breaks caused by replication fork collapse or shortening of telomeres (SAINI et al 2013;BUZOVETSKY et al 2017). During BIR, Rad51-mediated invasion of ssDNA into a homologous template creates a migrating bubble, resulting in conservative synthesis of a nascent DNA duplex (DONNIANNI AND SYMINGTON 2013;KRAMARA et al 2018).…”