Loss of Pol32 in Drosophila melanogaster Causes Chromosome Instability and Suppresses Variegation

Tritto, Patrizia; Palumbo, Valeria; Micale, Lucia; Marzulli, Marco; Bozzetti, Maria Pia; Specchia, Valeria; Palumbo, G.; Pimpinelli, Sergio; Berloco, Maria

doi:10.1371/journal.pone.0120859

Cited by 8 publications

(11 citation statements)

References 61 publications

(65 reference statements)

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“…First, it might work alongside or in combination with POL32 to promote Pol  processivity. Flies lacking POL32 are also sensitive to HU (our unpublished data) and eggs lacking POL32 have severe nuclear division defects during embryogenesis and fail to develop to larval stages (TRITTO et al 2015). In addition, pol32 mutant embryonic defects cannot be rescued by paternal expression of POL32 (TRITTO et al 2015), similar to the pif1 mutant phenotype.…”

Section: Drosophila Pif1 Is Important During Early Embryo Developmentmentioning

confidence: 76%

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

Kocak

Dykstra

Németh

et al. 2019

Preprint

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PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acidprotein 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 homologous recombination and 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 to promote efficient DNA synthesis during times of replication stress and acts independently of POL32 to promote synthesis during doublestrand gap repair.

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Section: Drosophila Pif1 Is Important During Early Embryo Developmentmentioning

confidence: 76%

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

Kocak

Dykstra

Németh

et al. 2019

Preprint

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“…Mutations in DNA replication factors lead to defects in position-effect variegation in flies (36)(37)(38) and silencing defects at telomeres and silent mating loci in yeast (39)(40)(41). Likewise, perturbations of cellular acyl pools in yeast by genetic inactivation orthologs of Acaca (42) and Acss1 (43) cause global alterations in histone acetylation and genomewide expression.…”

Section: Discussionmentioning

confidence: 99%

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

Sripathy

Leko

Adrianse

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.XIST | X inactivation | MeCP2 | Rett syndrome | BMP/TGF-β

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“…First, it might work alongside or in combination with POL32 to promote Pol d processivity. Flies lacking POL32 are also sensitive to hydroxyurea (D. Kane and M. McVey, unpublished data) and eggs lacking POL32 have severe nuclear division defects during embryogenesis and fail to develop to larval stages (Tritto et al 2015). In addition, pol32 mutant embryonic defects cannot be rescued by paternal expression of POL32 (Tritto et al 2015), similar to the pif1 mutant phenotype.…”

Section: A Critical Role For Drosophila Pif1 During Replication Stressmentioning

confidence: 97%

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

Kocak

Dykstra²,

Németh³

et al. 2019

Genetics

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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 ...

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Loss of Pol32 in Drosophila melanogaster Causes Chromosome Instability and Suppresses Variegation

Cited by 8 publications

References 61 publications

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

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

Screen for reactivation of MeCP2 on the inactive X chromosome identifies the BMP/TGF-β superfamily as a regulator of XIST expression

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

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