The Drosophila melanogaster PIF1 helicase promotes survival during replication stress and processive DNA synthesis during double-strand gap repair

AbstractPIF1 is a 5’ to 3’ 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.

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

Genetics ◽

10.1534/genetics.119.302665 ◽

2019 ◽

Vol 213 (3) ◽

pp. 835-847 ◽

Cited By ~ 1

Author(s):

Ece Kocak ◽

Sarah Dykstra ◽

Alexandra Nemeth ◽

Catherine G. Coughlin ◽

Kasey Rodgers ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Dna Synthesis ◽

Replication Stress ◽

Pif1 Helicase

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Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.103 ◽

2001 ◽

Vol 157 (1) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Janet R Mullen ◽

Vivek Kaliraman ◽

Samer S Ibrahim ◽

Steven J Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Stability ◽

Protein Complexes ◽

Ring Finger ◽

Dna Helicase ◽

Open Reading Frames ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Helicases ◽

Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells

Blood Cancer Journal ◽

10.1038/s41408-021-00531-2 ◽

2021 ◽

Vol 11 (7) ◽

Author(s):

Ruochen Jia ◽

Leon Kutzner ◽

Anna Koren ◽

Kathrin Runggatscher ◽

Peter Májek ◽

...

Keyword(s):

High Throughput ◽

Drug Screening ◽

Synthetic Lethality ◽

Myeloproliferative Neoplasms ◽

Hematopoietic Cells ◽

Replication Stress ◽

Phase Cell ◽

Nuclear Staining ◽

Wild Type ◽

High Throughput Drug Screening

AbstractMutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

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A Requirement for Recombinational Repair in Saccharomyces cerevisiae Is Caused by DNA Replication Defects of mec1 Mutants

Genetics ◽

10.1093/genetics/153.2.595 ◽

1999 ◽

Vol 153 (2) ◽

pp. 595-605 ◽

Cited By ~ 2

Author(s):

Bradley J Merrill ◽

Connie Holm

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Synthetic Lethality ◽

Velocity Sedimentation ◽

Recombinational Repair ◽

Repair Pathway ◽

Dna Breaks ◽

Single Stranded Dna ◽

Double Stranded Dna

Abstract To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.

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Augmenting the cytotoxicity of oleic acid-protein complexes: Potential of target-specific antibodies

Biochimie ◽

10.1016/j.biochi.2017.03.013 ◽

2017 ◽

Vol 137 ◽

pp. 139-146 ◽

Cited By ~ 1

Author(s):

Mehboob Hoque ◽

Jyoti Gupta ◽

M. Saleemuddin

Keyword(s):

Oleic Acid ◽

Protein Complexes ◽

Specific Antibodies ◽

Acid Protein

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Stimulation of DNA Synthesis by Mouse DNA Helicase B in a DNA Replication System Containing Eukaryotic Replication Origins

Biochemistry ◽

10.1021/bi00024a016 ◽

1995 ◽

Vol 34 (24) ◽

pp. 7913-7922 ◽

Cited By ~ 10

Author(s):

Ken Matsumoto ◽

Masayuki Seki ◽

Chikahide Masutani ◽

Shusuke Tada ◽

Takemi Enomoto ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Helicase ◽

Replication Origins ◽

Replication System ◽

Stimulation Of

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Uncovering an allosteric mode of action for a selective inhibitor of human Bloom syndrome protein

10.1101/2020.11.30.403493 ◽

2020 ◽

Author(s):

X. Chen ◽

Y. Ali ◽

C.E.L. Fisher ◽

R. Arribas-Bosacoma ◽

M.B. Rajasekaran ◽

...

Keyword(s):

Synthetic Lethality ◽

Specific Inhibitor ◽

Bloom Syndrome ◽

Dna Helicase ◽

Holliday Junctions ◽

Dna Structures ◽

Recq Helicases ◽

Promising Target ◽

Opening And Closing ◽

Syndrome Protein

ABSTRACTBLM (Bloom syndrome protein) is a RECQ-family helicase involved in the dissolution of complex DNA structures and repair intermediates. Synthetic lethality analysis implicates BLM as a promising target in a range of cancers with defects in the DNA damage response, however selective small molecule inhibitors of defined mechanism are currently lacking. Here we identify and characterise a specific inhibitor of BLM’s ATPase-coupled DNA helicase activity, by allosteric trapping of a DNA-bound translocation intermediate. Crystallographic structures of BLM-DNA-ADP-inhibitor complexes identify a hitherto unknown interdomain interface, whose opening and closing are integral to translocation of ssDNA, and which provides a highly selective pocket for drug discovery. Comparison with structures of other RECQ helicases provides a model for branch migration of Holliday junctions by BLM.

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DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2024258118 ◽

2021 ◽

Vol 118 (17) ◽

pp. e2024258118

Author(s):

Nanda Kumar Jegadesan ◽

Dana Branzei

Keyword(s):

Dna Repair ◽

Cancer Cells ◽

Drug Hypersensitivity ◽

Replication Stress ◽

Strand Break ◽

Parp Inhibitors ◽

Dna Helicase ◽

Brca1 And Brca2 ◽

Focus Formation ◽

Chemotherapy Drug

DDX11 encodes an iron–sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug–resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

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