Role and Regulation of Pif1 Family Helicases at the Replication Fork

Malone, Emory G.; Thompson, Matthew D.; Byrd, Alicia K.

doi:10.3390/ijms23073736

Cited by 7 publications

(8 citation statements)

References 182 publications

(325 reference statements)

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“…When dia2Δ cells progress through S-phase, old CMG complexes from the previous cell cycle are removed by an unknown pathway. One candidate factor for such a pathway is the Rrm3 DNA helicase, which together with its paralogue Pif1 is known to help replication forks pass a range of roadblocks on chromatin (Bochman et al , 2010; Malone et al , 2022; Muellner & Schmidt, 2020), such as tightly bound protein-DNA complexes including Mcm2-7 double hexamers (Hill et al , 2020) or stable complexes at tRNA promoters or telomeres (Ivessa et al , 2003; Ivessa et al , 2002), or stable DNA structures such as G-quadruplexes (Kumar et al , 2021; Paeschke et al , 2011; Ribeyre et al , 2009; Williams et al , 2023). Previous work showed that both Rrm3 and Pif1 are important for the viability of dia2Δ cells (Blake et al ., 2006; Morohashi et al ., 2009), and we found that dia2Δ cells lacking both Rrm3 and Pif1 are inviable (Fig 5A).…”

Section: Resultsmentioning

confidence: 99%

CMG helicase disassembly is essential and driven by two pathways in budding yeast

Rivera,

Deegan,

Labib

2024

Preprint

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The CMG helicase is the metastable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, indicating that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

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Section: Resultsmentioning

confidence: 99%

CMG helicase disassembly is essential and driven by two pathways in budding yeast

Rivera,

Deegan,

Labib

2024

Preprint

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“…RAD51 is a hallmark protein of homologous recombination [56,57]. BIR is a form of homologous recombination that involves the repair of single-ended DNA DSBs on folded replication forks [9]. PIF1 readily localizes to replication-associated fragile sites, inducing BIR [25].…”

Section: Discussionmentioning

confidence: 99%

“…PIF1 helicase (5 →3 DNA helicase), a member of the helicase superfamily 1, translocates along singlestranded DNA and unwinds double-stranded DNA in the 5 →3 direction [8]. PIF1 helicases localize to and facilitate the advancement of replication forks [9]. Additionally, PIF1 facilitates Okazaki fragment synthesis [10].…”

Section: Introductionmentioning

confidence: 99%

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Downregulation of PIF1 induce DNA damage and inhibit ovarian cancer cell proliferation via RAD51

Zhou,

Hua,

Sun

et al. 2024

Preprint

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Background PIF1 helicase (5ʹ→3ʹ DNA helicase) is a member of helicase superfamily 1. It has unwinding activity and plays a crucial role in maintaining genome stability and coordinating DNA damage repair processes. Overexpression of PIF1 is common in several cancers; however, its role in ovarian cancer remains unclear. This study aimed to elucidate the regulatory role of PIF1 in ovarian cancer and explore its mechanism. Results Analysis of patient samples and public database datasets revealed a negative correlation between PIF1 overexpression and the overall survival rate of the patients. We found through molecular biology experiments and xenograft tumor models in nude mice that CRISPR/Cas9-mediated PIF1 partial knockdown in ovarian cancer cell lines significantly inhibited proliferation and clonogenicity, promoted senescence, and induced G2 cell cycle arrest. Moreover, PIF1 partial deficiency enhanced DNA damage in ovarian cancer cells, particularly sensitive to cisplatin. RAD51 serves as a central scaffold protein for homologous recombination repair and is crucial for timely and accurate DNA repair. We observed that PIF1 partial knockdown resulted in significant reduction of RAD51 in ovarian cancer cells. Notably, RAD51 overexpression in PIF1 partially deficient ovarian cancer cells rescued cell proliferation and DNA damage by increasing PIF1 expression. Immunofluorescence revealed the co-localization of EGFP-PIF1 and RAD51 in the cell nucleus, suggesting that the interaction between PIF1 and RAD51 may regulate the DNA damage response and cell survival in ovarian cancer cells. Conclusions Our study revealed that PIF1 is a druggable target for inducing DNA damage in ovarian cancer cells and provides insights into the potential synergistic mechanisms of action between PIF1 and RAD51 in ovarian cancer therapy.

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“…Pif1 helicase has been identified in S. cerevisiae as a protein with multiple functions. Pif1 is involved in DNA repair and is necessary for the maintenance of mitochondrial and nuclear genomes integrity, in the regulation of telomerase activity and in the replication fork progression (reviewed in [ 3 , 4 , 5 , 6 ]). Many functions of Pif1 could be explained by its specific activity towards G-quadruplexes (G4), a particularly stable structure of NA which can be adopted by Guanine-rich sequences [ 7 ].…”

Section: Introductionmentioning

confidence: 99%

Structural Studies of Pif1 Helicases from Thermophilic Bacteria

Réty

Zhang

et al. 2023

Microorganisms

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Pif1 proteins are DNA helicases belonging to Superfamily 1, with 5′ to 3′ directionality. They are conserved from bacteria to human and have been shown to be particularly important in eukaryotes for replication and nuclear and mitochondrial genome stability. However, Pif1 functions in bacteria are less known. While most Pif1 from mesophilic bacteria consist of the helicase core with limited N-terminal and C-terminal extensions, some Pif1 from thermophilic bacteria exhibit a C-terminal WYL domain. We solved the crystal structures of Pif1 helicase cores from thermophilic bacteria Deferribacter desulfuricans and Sulfurihydrogenibium sp in apo and nucleotide bound form. We show that the N-terminal part is important for ligand binding. The full-length Pif1 helicase was predicted based on the Alphafold algorithm and the nucleic acid binding on the Pif1 helicase core and the WYL domain was modelled based on known crystallographic structures. The model predicts that amino acids in the domains 1A, WYL, and linker between the Helicase core and WYL are important for nucleic acid binding. Therefore, the N-terminal and C-terminal extensions may be necessary to strengthen the binding of nucleic acid on these Pif1 helicases. This may be an adaptation to thermophilic conditions.

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Role and Regulation of Pif1 Family Helicases at the Replication Fork

Cited by 7 publications

References 182 publications

CMG helicase disassembly is essential and driven by two pathways in budding yeast

CMG helicase disassembly is essential and driven by two pathways in budding yeast

Downregulation of PIF1 induce DNA damage and inhibit ovarian cancer cell proliferation via RAD51

Structural Studies of Pif1 Helicases from Thermophilic Bacteria

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