scholarly journals PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

2008 ◽  
Vol 183 (7) ◽  
pp. 1203-1212 ◽  
Author(s):  
Kazuto Sugimura ◽  
Shin-ichiro Takebayashi ◽  
Hiroshi Taguchi ◽  
Shunichi Takeda ◽  
Katsuzumi Okumura
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Parp Inhibitor ◽  
Small Interfering Rnas ◽  
Parp 1 ◽  
Dt40 Cells ◽  
Damaged Dna

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.

scholarly journals RAD18 and Poly(ADP-Ribose) Polymerase Independently Suppress the Access of Nonhomologous End Joining to Double-Strand Breaks and Facilitate Homologous Recombination-Mediated Repair

2007 ◽  
Vol 27 (7) ◽  
pp. 2562-2571 ◽  
Author(s):  
Alihossein Saberi ◽  
Helfrid Hochegger ◽  
David Szuts ◽  
Li Lan ◽  
Akira Yasui ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dt40 Cells

ABSTRACT The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.

scholarly journals PARP Inhibitor Olaparib Causes No Potentiation of the Bleomycin Effect in VERO Cells, Even in the Presence of Pooled ATM, DNA-PK, and LigIV Inhibitors

2020 ◽  
Vol 21 (21) ◽  
pp. 8288
Author(s):  
Valentina Perini ◽  
Michelle Schacke ◽  
Pablo Liddle ◽  
Salomé Vilchez-Larrea ◽  
Deborah J. Keszenman ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Synthetic Lethality ◽  
Excision Repair ◽  
Parp Inhibitor ◽  
Genotoxic Stress ◽  
Vero Cells ◽  
Limiting Factor ◽  
Parp Activation ◽  
Alternative Hypotheses

Poly(ADP-ribosyl)polymerase (PARP) synthesizes poly(ADP-ribose) (PAR), which is anchored to proteins. PAR facilitates multiprotein complexes’ assembly. Nuclear PAR affects chromatin’s structure and functions, including transcriptional regulation. In response to stress, particularly genotoxic stress, PARP activation facilitates DNA damage repair. The PARP inhibitor Olaparib (OLA) displays synthetic lethality with mutated homologous recombination proteins (BRCA-1/2), base excision repair proteins (XRCC1, Polβ), and canonical nonhomologous end joining (LigIV). However, the limits of synthetic lethality are not clear. On one hand, it is unknown whether any limiting factor of homologous recombination can be a synthetic PARP lethality partner. On the other hand, some BRCA-mutated patients are not responsive to OLA for still unknown reasons. In an effort to help delineate the boundaries of synthetic lethality, we have induced DNA damage in VERO cells with the radiomimetic chemotherapeutic agent bleomycin (BLEO). A VERO subpopulation was resistant to BLEO, BLEO + OLA, and BLEO + OLA + ATM inhibitor KU55933 + DNA-PK inhibitor KU-0060648 + LigIV inhibitor SCR7 pyrazine. Regarding the mechanism(s) behind the resistance and lack of synthetic lethality, some hypotheses have been discarded and alternative hypotheses are suggested.

scholarly journals PARP-1 Activation Directs FUS to DNA Damage Sites to Form PARG-Reversible Compartments Enriched in Damaged DNA

Cell Reports ◽  
2019 ◽  
Vol 27 (6) ◽  
pp. 1809-1821.e5 ◽  
Author(s):  
Anastasia S. Singatulina ◽  
Loic Hamon ◽  
Maria V. Sukhanova ◽  
Bénédicte Desforges ◽  
Vandana Joshi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Parp 1 ◽  
Damaged Dna

scholarly journals Homologous Recombination Is the Principal Pathway for the Repair of DNA Damage Induced by Tirapazamine in Mammalian Cells

2008 ◽  
Vol 68 (1) ◽  
pp. 257-265 ◽  
Author(s):  
James W. Evans ◽  
Sophia B. Chernikova ◽  
Lisa A. Kachnic ◽  
Judit P. Banath ◽  
Olivier Sordet ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Principal Pathway

The roles of hypoxia, PTEN, and Rad51 in mediating metastatic prostate cancer cells' responses to PARP inhibitor and topoisomerase 1 inhibitor.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13564-e13564
Author(s):  
Jingsong Zhang ◽  
Minghui Wu ◽  
Xue Wang
Keyword(s):  
Prostate Cancer ◽  
Dna Damage ◽  
Topoisomerase I ◽  
Metastatic Prostate Cancer ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Single Agent ◽  
Study Results

e13564 Background: With the recent success of poly (ADP-ribose) polymerase inhibitor (PARPi) in the treatment of BRCA1 or BRCA2 mutated cancers, there is increasing interest to explore synthetic lethality in cancers with defective DNA repair pathways. Rad51 is an essential protein in the homologous recombination repair (HRR) of DNA double strand breaks. Previous studies with non metastatic prostate cancer (mCaP) cells have reported low Rad51 levels in cells with loss of PTEN or under hypoxia, which then led to their sensitivity to PARPi. Given intra tumor hypoxia and loss of PTEN is common in mCaP, we test PAPRi, ABT888 and DNA damaging topoisomerase I inhibitor, CPT11, either alone or in combination in mCaP preclinical models. Methods: mCaP cell lines with functional PTEN (DU145) and loss of PTEN (PC3) were grew under normoxia (21% O2) or hypoxia (0.2% O2). DNA damage, HRR, apoptosis were assessed with comet assay, western blot, immunofluorescence and flowcytometry. The regulation of RAD51 was studied with quantitative RT-PCR and RAD51 promoter reporter assay. PC3 xenograft was used for in vivo study. Results: Despite of its low levels of expression under hypoxia, up regulation of Rad51 was observed soon after treating hypoxic PC3 and Du145 cells with ABT888 or SN38, an active metabolite of CPT11. Such Rad51 up regulation led to less DNA damage and apoptosis under hypoxia compared to normoxia. Inhibiting RAD51 expression with siRNA overcame PC3 and Du145’s resistance to SN38. Furthermore, ABT888 enhanced the activities of SN38 as detected by clonogenic assay and flowcytometry under both normoxia and hypoxia. Consistent with the in vitro data, ABT888 by itself had limited anti-tumor activities despite the loss of PTEN in PC3 xenografts. The anti-tumor activity of single agent CPT11 was significantly improved with the ABT888 and CPT11 combination (P<0.008). Conclusions: neither loss of PTEN nor hypoxia sensitized mCaP cells to PARPi or DNA damaging drugs. Such resistance under hypoxia was at least partly due to up regulation of Rad51. Combining ABT888 with CPT11 overcame the resistance to CPT11 under hypoxia and enhanced its anti-tumor activities both in vitro and in vivo.

scholarly journals Role for RAD18 in Homologous Recombination in DT40 Cells

2006 ◽  
Vol 26 (21) ◽  
pp. 8032-8041 ◽  
Author(s):  
Dávid Szüts ◽  
Laura J. Simpson ◽  
Sarah Kabani ◽  
Mitsuyoshi Yamazoe ◽  
Julian E. Sale
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Early Stage ◽  
Current Evidence ◽  
Recombination Reaction ◽  
Immunoglobulin Gene ◽  
Postreplication Repair ◽  
Strand Breaks ◽  
Dt40 Cells

ABSTRACT RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

PARP inhibitors for cancer therapy

2005 ◽  
Vol 7 (4) ◽  
pp. 1-20 ◽  
Author(s):  
Nicola J. Curtin
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Topoisomerase I ◽  
Alkylating Agents ◽  
Parp Inhibitors ◽  
Ionising Radiation ◽  
Parp Inhibition ◽  
Tumour Regression ◽  
Dna Breaks ◽  
Parp 1

Poly(ADP-ribose) polymerase 1 (PARP-1) is a zinc-finger DNA-binding enzyme that is activated by binding to DNA breaks. Poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 converts DNA damage into intracellular signals that activate either DNA repair by the base-excision pathway or cell death. A family of 18 PARPs has been identified, but only the most abundant, PARP-1 and PARP-2, which are both nuclear enzymes, are activated by DNA damage. PARP inhibitors of ever-increasing potency have been developed in the 40 years since the discovery of PARP-1, both as tools for the investigation of PARP-1 function and as potential modulators of DNA-repair-mediated resistance to cytotoxic therapy. Owing to the high level of homology between the catalytic domains of PARP-1 and PARP-2, the inhibitors probably affect both enzymes. Convincing biochemical evidence, which has been corroborated by genetic manipulation of PARP-1 activity, shows that PARP inhibition is associated with increased sensitivity to DNA-alkylating agents, topoisomerase I poisons and ionising radiation. Novel PARP inhibitors of sufficient potency and suitable pharmacokinetic properties to allow evaluation in animal models have been shown to enhance the antitumour activity of temozolomide (a DNA-methylating agent), topoisomerase poisons and ionising radiation; indeed, the combination with temozolomide resulted in complete tumour regression in two independent studies. The combination of a PARP inhibitor and temozolomide is currently undergoing clinical evaluation for the first time.

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