scholarly journals ATAD5 deficiency alters DNA damage metabolism and sensitizes cells to PARP inhibition

2020 ◽  
Vol 48 (9) ◽  
pp. 4928-4939 ◽  
Author(s):  
Sara Giovannini ◽  
Marie-Christine Weller ◽  
Hana Hanzlíková ◽  
Tetsuya Shiota ◽  
Shunichi Takeda ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Replication Forks ◽  
Replication Factor C ◽  
Knock Out ◽  
Endometrial Cancers ◽  
Factor C

Abstract Replication factor C (RFC), a heteropentamer of RFC1-5, loads PCNA onto DNA during replication and repair. Once DNA synthesis has ceased, PCNA must be unloaded. Recent findings assign the uloader role primarily to an RFC-like (RLC) complex, in which the largest RFC subunit, RFC1, has been replaced with ATAD5 (ELG1 in Saccharomyces cerevisiae). ATAD5-RLC appears to be indispensable, given that Atad5 knock-out leads to embryonic lethality. In order to learn how the retention of PCNA on DNA might interfere with normal DNA metabolism, we studied the response of ATAD5-depleted cells to several genotoxic agents. We show that ATAD5 deficiency leads to hypersensitivity to methyl methanesulphonate (MMS), camptothecin (CPT) and mitomycin C (MMC), agents that hinder the progression of replication forks. We further show that ATAD5-depleted cells are sensitive to poly(ADP)ribose polymerase (PARP) inhibitors and that the processing of spontaneous oxidative DNA damage contributes towards this sensitivity. We posit that PCNA molecules trapped on DNA interfere with the correct metabolism of arrested replication forks, phenotype reminiscent of defective homologous recombination (HR). As Atad5 heterozygous mice are cancer-prone and as ATAD5 mutations have been identified in breast and endometrial cancers, our finding may open a path towards the therapy of these tumours.

scholarly journals Synthetic lethality between BRCA1 deficiency and poly(ADP-ribose) polymerase inhibition is modulated by processing of endogenous oxidative DNA damage

2019 ◽  
Vol 47 (17) ◽  
pp. 9132-9143 ◽  
Author(s):  
Sara Giovannini ◽  
Marie-Christine Weller ◽  
Simone Repmann ◽  
Holger Moch ◽  
Josef Jiricny
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Synthetic Lethality ◽  
Oxygen Metabolism ◽  
Parp Inhibitors ◽  
Parp Inhibition ◽  
Strand Breaks ◽  
Hypoxic Conditions ◽  
Single Strand Breaks ◽  
Clinical Resistance

Abstract Poly(ADP-ribose) polymerases (PARPs) facilitate the repair of DNA single-strand breaks (SSBs). When PARPs are inhibited, unrepaired SSBs colliding with replication forks give rise to cytotoxic double-strand breaks. These are normally rescued by homologous recombination (HR), but, in cells with suboptimal HR, PARP inhibition leads to genomic instability and cell death, a phenomenon currently exploited in the therapy of ovarian cancers in BRCA1/2 mutation carriers. In spite of their promise, resistance to PARP inhibitors (PARPis) has already emerged. In order to identify the possible underlying causes of the resistance, we set out to identify the endogenous source of DNA damage that activates PARPs. We argued that if the toxicity of PARPis is indeed caused by unrepaired SSBs, these breaks must arise spontaneously, because PARPis are used as single agents. We now show that a significant contributor to PARPi toxicity is oxygen metabolism. While BRCA1-depleted or -mutated cells were hypersensitive to the clinically approved PARPi olaparib, its toxicity was significantly attenuated by depletion of OGG1 or MYH DNA glycosylases, as well as by treatment with reactive oxygen species scavengers, growth under hypoxic conditions or chemical OGG1 inhibition. Thus, clinical resistance to PARPi therapy may emerge simply through reduced efficiency of oxidative damage repair.

scholarly journals Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

1998 ◽  
Vol 18 (9) ◽  
pp. 5485-5491 ◽  
Author(s):  
Toshiyasu Shimomura ◽  
Seiko Ando ◽  
Kunihiro Matsumoto ◽  
Katsunori Sugimoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Small Subunit ◽  
Dna Damage Checkpoint ◽  
Physical Interaction ◽  
Replication Factor C ◽  
Functional Interaction ◽  
Dna Damaging Agents ◽  
Replication Block ◽  
Factor C

ABSTRACT The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation ofRAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24, like Rfc5, is required for the regulation of Rad53 phosphorylation in response to DNA damage. The Rad24 protein, which is structurally related to the RFC subunits, interacts physically with RFC subunits Rfc2 and Rfc5 and cosediments with Rfc5. Although therad24Δ mutation alone does not cause a defect in the replication block checkpoint, it does enhance the defect inrfc5-1 mutants. Furthermore, overexpression ofRAD24 suppresses the rfc5-1 defect in the replication block checkpoint. Taken together, our results demonstrate a physical and functional interaction between Rad24 and Rfc5 in the checkpoint pathways.

scholarly journals The Large Subunit of Replication Factor C (Rfclp/Cdc44p) Is Required for DNA Replication and DNA Repair in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 65-78 ◽  
Author(s):  
Michael A McAlear ◽  
K Michelle Tuffo ◽  
Connie Holm
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Dna Replication ◽  
Uv Radiation ◽  
Large Subunit ◽  
Restrictive Temperature ◽  
Replication Factor C ◽  
Replication Factor ◽  
Factor C

We used genetic and biochemical techniques to characterize the phenotypes associated with mutations affecting the large subunit of replication factor C (Cdc44p or Rfc1p) in Saccharomyces cerevisiae. We demonstrate that Cdc44p is required for both DNA replication and DNA repair in vivo. Cold-sensitive cdc44 mutants experience a delay in traversing S phase at the restrictive temperature following alpha factor arrest; although mutant cells eventually accumulate with a G2/M DNA content, they undergo a cell cycle arrest and initiate neither mitosis nor a new round of DNA synthesis. cdc44 mutants also exhibit an elevated level of spontaneous mutation, and they are sensitive both to the DNA damaging agent methylmethane sulfonate and to exposure to UV radiation. After exposure to UV radiation, cdc44 mutants at the restrictive temperature contain higher levels of single-stranded DNA breaks than do wild-type cells. This observation is consistent with the hypothesis that Cdc44p is involved in repairing gaps in the DNA after the excision of damaged bases. Thus, Cdc44p plays an important role in both DNA replication and DNA repair in vivo.

CBIO-22. PARP INHIBITION SYNERGIZES WITH DNA DAMAGING DRUGS IN PEDIATRIC CNS TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi31-vi31
Author(s):  
Anna Laemmerer ◽  
Dominik Kirchhofer ◽  
Sibylle Madlener ◽  
Daniela Loetsch-Gojo ◽  
Carola Jaunecker ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Predictive Biomarkers ◽  
High Grade Glioma ◽  
Parp Inhibitors ◽  
Cns Tumors ◽  
Parp Inhibition ◽  
Tumor Subtypes ◽  
Synergistic Cytotoxicity ◽  
Anti Cancer

Abstract BACKGROUND Central nervous system (CNS) tumors are the second most common childhood cancer. Despite innovations in surgery and chemo-/radiotherapy, CNS tumors remain the major cause of cancer-related death in children. Previous sequencing analyses in a pediatric cancer cohort identified BRCA and DSB repair signatures as potentially targetable events. Based on these findings, we propose the use of PARP inhibitors (PARPi) for aggressive CNS tumor subtypes, including high-grade glioma (HGG), medulloblastoma (MB) and ependymoma (EPN). METHODS We tested multiple PARPi in tumor cell lines (n=8) as well as primary patient-derived models (n=11) of pediatric HGG, MB, EPN and atypical teratoid/rhabdoid tumors (ATRTs). Based on PARPi sensitivity, selected models were further exposed to a combination of PARPi and DNA-damaging/modifying agents. The mode of action was investigated using Western blot and flow cytometry. RESULTS We show that a fraction of pediatric MB, EPN and ATRT demonstrate sensitivity towards PARP inhibition, which is paralleled by susceptibility to the DNA damaging drugs cisplatin and irinotecan. Interestingly, talazoparib, the most potent PARPi, showed synergistic cytotoxicity with DNA-damaging/modifying drugs. In addition, cell cycle blockade and increased DNA damage combined with reduced DNA repair signaling, such as activation of the ATR/Chk1 pathway were observed. Corroboratively, talazoparib exhibited a synergistic anti-cancer effect in combination with inhibitors of ATR, a major regulator of DNA damage response. CONCLUSION/OUTLOOK To sum up, we demonstrate that PARP inhibition synergizes with DNA damaging anti-cancer compounds or DNA repair inhibitors and, thus, represents a promising therapeutic strategy for a defined subgroup of pediatric high-risk CNS tumors patients. More in depth characterization of the underlying molecular events will most likely allow the identification of predictive biomarkers for most efficient implementation of this strategy into clinical application.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex

1992 ◽  
Vol 12 (1) ◽  
pp. 155-163 ◽  
Author(s):  
K Fien ◽  
B Stillman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Replication Complex ◽  
Leading Strand ◽  
Factor C ◽  
Sv40 Dna

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.

scholarly journals Brewer’s Spent Grains Protects against Oxidative DNA Damage in Saccharomyces cerevisiae

2017 ◽  
Vol 9 (7) ◽  
pp. 12
Author(s):  
Manuela M. Moreira ◽  
Daniel O. Carvalho ◽  
Rui Oliveira ◽  
Björn Johansson ◽  
Luís F. Guido
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Flow Cytometry ◽  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Yeast Cells ◽  
Protective Effects ◽  
Genotoxic Effects ◽  
Barley Malt ◽  
Fold Reduction ◽  
Spent Grain

Brewer’s spent grain (BSG), obtained from barley malt during brewing, contains high amounts of phenolic acids, predominantly ferulic and p-coumaric acids. The protective effects of BSG extracts against oxidative DNA damage induced by H2O2 in Saccharomyces cerevisiae cells were investigated using an optimized yeast comet assay and flow cytometry. The results indicated that BSG extracts from black malt exhibited a 5-fold reduction in the genotoxic effects of H2O2, compared to the 2-fold decrease by the BSG extracts from pilsen malts. Flow cytometry analysis with dichlorofluorescein diacetate demonstrated that the intracellular oxidation of S. cerevisiae is also reduced to approximately 50% in the presence of 20-fold diluted BSG extracts. BSG extracts obtained from pilsen and black malt types exert dose-dependent protective properties against the genotoxic effects induced by ROS and decrease intracellular oxidation of yeast cells.

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