scholarly journals DDB2 promotes chromatin decondensation at UV-induced DNA damage

2012 ◽  
Vol 197 (2) ◽  
pp. 267-281 ◽  
Author(s):  
Martijn S. Luijsterburg ◽  
Michael Lindh ◽  
Klara Acs ◽  
Mischa G. Vrouwe ◽  
Alex Pines ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Chromatin Structure ◽  
Large Scale ◽  
Excision Repair ◽  
Adenosine Diphosphate ◽  
Dna Lesions ◽  
Genome Recognition ◽  
Core Histones ◽  
Steady State Levels

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.

scholarly journals Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

2008 ◽  
Vol 28 (24) ◽  
pp. 7402-7413 ◽  
Author(s):  
Sergey Alekseev ◽  
Martijn S. Luijsterburg ◽  
Alex Pines ◽  
Bart Geverts ◽  
Pierre-Olivier Mari ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dynamic Binding ◽  
Damage Site ◽  
Damage Recognition ◽  
Mutagenic Effects ◽  
Damaged Dna

ABSTRACT Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evi Goulielmaki ◽  
Maria Tsekrekou ◽  
Nikos Batsiotos ◽  
Mariana Ascensão-Ferreira ◽  
Eleftheria Ledaki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Splicing ◽  
Excision Repair ◽  
Intron Retention ◽  
Dna Lesions ◽  
Splicing Factor ◽  
Loop Formation ◽  
Rna Targets ◽  
Underlying Mechanisms

AbstractRNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

Genomic profiling of biliary tract cancers to reveal clinically actionable genes and therapeutic biomarkers.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e16187-e16187
Author(s):  
Yang Shao ◽  
Qiuxiang Ou ◽  
Zhenhao Fang ◽  
Rui Liu ◽  
Hua Bao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Genomic Instability ◽  
Biliary Tract ◽  
Large Scale ◽  
Excision Repair ◽  
State Transitions ◽  
Pathway Enrichment Analysis ◽  
Distal Cholangiocarcinoma ◽  
Tract Cancer

e16187 Background: Bile tract cancers are genetically and clinically heterogenous with a poor prognosis. Identifying novel biomarkers for targeted therapy is required to improve the clinical outcome of bile tract cancer patients. Methods: Tumor tissue samples of 482 Chinese biliary tract cancer (BTC) patients were genetically profiled using targeted next generation sequencing. Tumor mutation burden (TMB) was calculated by counting all nonsynonymous mutations per megabase of coding sequences. The R package ReactomePA was used in pathway enrichment analysis. Genomic instability was characterized by an in-house developed NGS-based Homologous Recombination Deficiency (HRD) panel and a HRD score was an unweighted sum of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) scores. Results: The BTC cohort consisted of 135 gallbladder cancer (GBC), 73 intrahepatic cholangiocarcinoma (iCCA), 18 distal cholangiocarcinoma (dCCA), 14 perihilar cholangiocarcinoma (pCCA), while the remaining 242 BTC patients of no specific subtype information. Most frequently mutated genes included TP53 (56%), KRAS (25%), ARID1A (17%), SMAD4 (11%), and CDKN2A (10%) . A preliminary pathway analysis revealed that mutations of DNA damage repair (DDR) pathway genes were enriched in the cohort ( p< 1e-10), accounting for over 70% of the patients, particularly in homologous recombination repair (HRR), Fanconi anemia (FA), mismatch repair (MMR), and base excision repair (BER) genes. More specifically, approximately 50% of the cohort carried at least one mutation of the HRR genes (43%) or MMR genes (14%). Patients with impaired MMR had increased microsatellite instability status (MSI) comparing to those with wildtype MMR (33% vs. 3.1%, p< 0.0001), and patients harboring HRR mutations demonstrated elevated genomic instability than those without such mutations (median HRD: 18 vs.14, p < 0.05), indicative of potential response to poly (ADP-ribose) polymerase (PARP) inhibitors and other DNA-damage agents. Furthermore, high TMB was found to be highly correlated with DDR gene alterations ( p =0.004). In addition, we observed higher mutation frequencies of BRCA1/2 genes (including somatic and germline) in GBCs in contrast to other BTC subtypes. Conclusions: We herein reported the genomic features of 482 Chinese BTC samples and highlighted the role of DDR pathways including HRR and MMR. These findings could be useful to establish treatment and diagnostic strategies for BTC patients based on genetic information.

scholarly journals Mutational signatures are jointly shaped by DNA damage and repair

2019 ◽  
Author(s):  
Nadezda V Volkova ◽  
Bettina Meier ◽  
Víctor González-Huici ◽  
Simone Bertolini ◽  
Santiago Gonzalez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Dna Lesions ◽  
Single Nucleotide Variants ◽  
Mutational Signatures ◽  
Experimental Conditions ◽  
Repair Deficiency ◽  
Dna Repair Deficiency ◽  
Mutation Spectra

AbstractMutations arise when DNA lesions escape DNA repair. To delineate the contributions of DNA damage and DNA repair deficiency to mutagenesis we sequenced 2,717 genomes of wild-type and 53 DNA repair defective C. elegans strains propagated through several generations or exposed to 11 genotoxins at multiple doses. Combining genotoxin exposure and DNA repair deficiency alters mutation rates or leads to unexpected mutation spectra in nearly 40% of all experimental conditions involving 9/11 of genotoxins tested and 32/53 genotypes. For 8/11 genotoxins, signatures change in response to more than one DNA repair deficiency, indicating that multiple genes and pathways are involved in repairing DNA lesions induced by one genotoxin. For many genotoxins, the majority of observed single nucleotide variants results from error-prone translesion synthesis, rather than primary mutagenicity of altered nucleotides. Nucleotide excision repair mends the vast majority of genotoxic lesions, preventing up to 99% of mutations. Analogous mutagenic DNA damage-repair interactions can also be found in cancers, but, except for rare cases, effects are weak owing to the unknown histories of genotoxic exposures and DNA repair status. Overall, our data underscore that mutation spectra are joint products of DNA damage and DNA repair and imply that mutational signatures computationally derived from cancer genomes are more variable than currently anticipated.

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Discovery and Optimisation of Bacterial Nitroreductases for Use in Anti-Cancer Gene Therapy

2021 ◽  
Author(s):  
◽  
Gareth Adrian Prosser
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Dna Lesions ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
E Coli ◽  
Anti Cancer

<p>Nitroaromatic prodrugs are biologically inert compounds that are attractive candidates for anti-cancer therapy by virtue of their ability to be converted to potent DNA alkylating agents by nitroreductase (NTR) enzymes. In gene-directed enzyme-prodrug therapy (GDEPT), NTR-encoding therapeutic transgenes are delivered specifically to tumour cells, whereupon their expression confers host cell sensitivity to subsequent systemic administration of a nitroaromatic prodrug. The most well studied NTR-GDEPT system involves reduction of the aziridinyl dinitrobenzamide prodrug CB1954 by the Escherichia coli NTR NfsB. However, low affinity of this enzyme for CB1954 has so far limited the clinical efficacy of this GDEPT combination. The research described in this thesis has primarily sought to address this limitation through identification and optimisation of novel NTR enzymes with improved nitroaromatic prodrug reductase activity. Efficient assessment of NTR activity from large libraries of candidate enzymes requires a rapid and reliable screening system. An E. coli-based assay was developed to permit indirect assessment of relative rates of prodrug reduction by over-expressed NTRs via measurement of SOS response induction resulting from reduced prodrug-induced DNA damage. Using this assay in concert with other in vitro and in vivo tests, more than 50 native bacterial NTRs of diverse sequence and origin were assessed for their ability to reduce a panel of clinically attractive nitroaromatic prodrugs. Significantly, a number of NTRs were identified, particularly in the family of enzymes homologous to the native E. coli NTR NfsA, which displayed substantially improved activity over NfsB with CB1954 and other nitroaromatic prodrugs as substrates. This work also examined the roles of E. coli DNA damage repair pathways in processing of adducts induced by various nitroaromatic prodrugs. Of particular interest, nucleotide excision repair was found to be important in the processing of DNA lesions caused by 4-, but not 2-nitro group reduction products of CB1954, which suggests that there are some parallels in the mechanisms of CB1954 adduct repair in E. coli and mammalian cells. Finally, a lead NTR candidate, YcnD from Bacillus subtilis, was selected for further activity improvement through site-directed mutagenesis of active site residues. Using SOS screening, a double-site mutant was identified with 2.5-fold improved activity over the wildtype enzyme in metabolism of the novel dinitrobenzamide mustard prodrug PR-104A. In conclusion, novel NTRs with substantially improved nitroaromatic prodrug reducing activity over previously documented enzymes were identified and characterised. These results hold significance not only for the field of NTR-GDEPT, but also for other biotechnological applications in which NTRs are becoming increasingly significant, including developmental studies, antibiotic discovery and bioremediation. Furthermore, the in vitro assays developed in this study have potential utility in the discovery and evolution of other GDEPT-relevant enzymes whose prodrug metabolism is associated with genotoxicity.</p>

scholarly journals Oxygen-Dependent Accumulation of Purine DNA Lesions in Cockayne Syndrome Cells

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1671 ◽  
Author(s):  
Marios G. Krokidis ◽  
Mariarosaria D’Errico ◽  
Barbara Pascucci ◽  
Eleonora Parlanti ◽  
Annalisa Masi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Cockayne Syndrome ◽  
Dna Lesions ◽  
Premature Aging ◽  
Neurological Features ◽  
Oxygen Tensions ◽  
Base Excision

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/106 nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage.

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