The Drosophila melanogaster PIF1 Helicase Promotes Survival During Replication Stress and Processive DNA Synthesis During Double-Strand Gap Repair

Abstract

PIF1 is a 59 to 39 DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair. KEYWORDS break-induced replication; polymerase; homologous recombination; DNA damage; replication fork collapse T HE Pif1 family helicases are 59 to 39 superfamily 1 helicases that are highly conserved in most eukaryotes and some bacteria and are critical for DNA replication, recombination, and repair (Bochman et al. 2010; Chung 2014; Byrd and Raney 2017). Although Pif1 family helicases possess a conserved single-stranded DNA (ssDNA)-dependent helicase domain, their N-and C-terminal domains differ significantly in size and sequence between organisms (Supplemental Material, Figure S1) (Lahaye et al. 1991; Boulé and Zakian 2006). Furthermore, the cellular functions of PIF1 homologs in eukaryotes are variably conserved. Because human PIF1 appears to act as a tumor suppressor but is also required for the survival of cancer cells (Gagou et al. 2014), a better understanding of its contribution to genome stability in diverse cellular and organismal contexts is needed. Much of what is currently known about Pif1 structure and function comes from studies in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. S. cerevisiae possesses two Pif1 orthologs, ScPif1 and ScRrm3, which localize to both the mitochondria and nucleus (Ivessa et al. 2000; Bochman et al. 2010). ScPif1 was first discovered in a genetic screen where its deficiency resulted in reduced mitochondrial DNA (mtDNA) recombination (Foury and Kolodynski 1983). Further studies revealed that ScPif1 slows down replication progression in mtDNA to prevent double-strand breaks (DSBs) and cooperates with base excision ...

Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster

“…However, it is also possible that repair proceeds through a break-induced replication (BIR) mechanism. Indeed, we have previously shown that the PIF1 DNA helicase, which is required for BIR in budding yeast [ 46 , 47 ] and promotes BIR in Drosophila [ 48 ], is needed for the efficient repair of a P{w a } -induced gap in the absence of the POL32 protein [ 49 ]. If a BIR-like mechanism is involved in gap repair in this system, then the helicases tested in the current study may also play a role in this type of repair.…”

Identifying PIF1 as a Potential Target of Wenxia Changfu Formula in Promoting Lung Cancer Cell Apoptosis: Bioinformatics Analysis and Biological Evidence

“…Finally, 21 oncogenes were identified, and we selected PIF1 to further confirm its biological function in A549 cells based on the highcontent screening results. PIF1 is a multifunctional helicase and plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication [26][27][28]. It is conserved in most eukaryotes and some prokaryotes [29,30].…”

Role and Regulation of Pif1 Family Helicases at the Replication Fork

“…Although it is possible that, like hPIF1, Rrm3 promotes resection across G4 sequences, this seems unlikely because Rrm3 has not been reported to have G4 DNA unfolding activity. Drosophila PIF1 is critical for genome maintenance and the survival of embryos exposed to the replication stalling agent hydroxyurea [ 76 ]. As G4 structures contribute to replication fork stalling, this suggests that Pif1 family helicases may also respond to replication fork stalling at G4 structures in addition to promoting replication through these structures.…”

“…Unlike canonical S-phase DNA synthesis, BIR involves conservative DNA synthesis by Polδ in a migrating D-loop [ 164 ]. Yeast, drosophila, and human cells depleted of Pif1 are deficient in BIR, but the depletion of Rrm3 does not affect BIR [ 76 , 142 , 165 ]. It is possible that two Pif1 molecules participate in BIR, with one at the leading edge of the migrating D-loop, which stimulates strand displacement synthesis by Polδ through its interaction with PCNA [ 39 ].…”