scholarly journals p53 Activation by Cr(VI): A Transcriptionally Limited Response Induced by ATR Kinase in S-Phase

2019 ◽  
Vol 172 (1) ◽  
pp. 11-22 ◽  
Author(s):  
Michal W Luczak ◽  
Casey Krawic ◽  
Anatoly Zhitkovich
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Target Genes ◽  
Cultured Cells ◽  
Excision Repair ◽  
S Phase ◽  
Human Cells ◽  
P53 Activation ◽  
Atr Kinase ◽  
Primary Human Cells

Abstract Cellular reduction of carcinogenic chromium(VI) causes several forms of Cr-DNA damage with different genotoxic properties. Chromate-treated cultured cells have shown a strong proapoptotic activity of the DNA damage-sensitive transcription factor p53. However, induction of p53 transcriptional targets by Cr(VI) in rodent lungs was weak or undetectable. We examined Cr(VI) effects on the p53 pathway in human cells with restored levels of ascorbate that acts as a principal reducer of Cr(VI) in vivo but is nearly absent in standard cell cultures. Ascorbate-restored H460 and primary human cells treated with Cr(VI) contained higher levels of p53 and its Ser15 phosphorylation, which were induced by ATR kinase. Cr(VI)-stimulated p53 phosphorylation occurred in S-phase by a diffusible pool of ATR that was separate from the chromatin-bound pool targeting DNA repair substrates at the sites of toxic mismatch repair (MMR) of Cr-DNA adducts. Even when more abundantly present than after exposure to the radiomimetic bleomycin, Cr(VI)-stabilized p53 showed a much more limited activation of its target genes in two types of primary human cells. No increases in mRNA were found for nucleotide excision repair factors and a majority of proapoptotic genes. A weak transcription activity of Cr(VI)-upregulated p53 was associated with its low lysine acetylation in the regulatory C-terminal domain, resulting from the inability of Cr(VI) to activate ATM in ascorbate-restored cells. Thus, p53 activation by ascorbate-metabolized Cr(VI) represents a limited genome-protective response that is defective in upregulation of DNA repair genes and proapoptotic transcripts for elimination of damaged cells.

AML1-ETO Inhibits DNA Repair Pathways and Promotes the Accumulation of DNA Damage and the Activation of p53.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1412-1412
Author(s):  
Ondrej Krejci ◽  
Mark Wunderlich ◽  
Junping Wei ◽  
Paul R. Andreassen ◽  
Thomas X. Lu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Gamma Irradiation ◽  
Malignant Transformation ◽  
Target Genes ◽  
Excision Repair ◽  
Genetic Alterations ◽  
P53 Pathway ◽  
Genetic Changes ◽  
Repair Pathways

Abstract Malignant transformation is a multistep process that is promoted by the accumulation of genetic changes over time. The contribution of individual genetic changes to the development of leukemia and the processes that induce these changes are not yet fully understood. The generation of the fusion gene AML1-ETO is thought to be an early event in the development of AML with t(8;21). Cells expressing AML1-ETO have been detected in samples from healthy newborns, as well as in patient samples analyzed long before the manifestation of AML. For these reasons it is assumed that a pre-leukemic clone expressing AML1-ETO requires additional genetic changes to complete the malignant transformation. However, the mechanism whereby these changes are acquired and the role that AML1-ETO plays in this process remain unclear. We used a model of human CD34+ cells expressing the AML1-ETO fusion protein to elucidate the role of AML1-ETO in leukemogenesis. AML1-ETO expressing (AE) cells displayed characteristics of a pre-leukemia, with enhanced self-renewal and NOD/SCID engraftment but no ability to induce malignancy in vivo. Analysis of gene expression using Affymetrix HG U133 Plus2.0 gene chips revealed that multiple genes important in various DNA repair pathways were suppressed in AE cells: OGG1, UNG, TDG, MBD4, POLB, POLE and FEN1 (Base Excision Repair), MRE11, RAD50, ATM, CHEK1 and CDC25A (ATM pathway), and FANCA, FANCL and BRCA2 (Fanconi anemia proteins). Treatment with DNA interstrand crosslinking agents mitomycin C or melphalan revealed a block in the S-phase of the cell cycle in AE cells at doses not affecting control cells. AE cells were also more sensitive to gamma irradiation compared to vector-transduced cells. In AE cultures, a gradual accumulation of cells with DNA damage was detected by immunofluorescence microscopy, using phosphorylated histone H2A.X as a marker. AE cells had a hyperactive p53 pathway with increased p53 protein levels, upregulation of its target genes (p21, TP53I3 and DAPK1), and increased apoptosis compared to control cultures, as might be expected in cells with chronic DNA damage. Incubation under low oxygen conditions prevented the accumulation of DNA damage and resulted in decreased expression of p53 target genes (TP53I3 and DAPK1), indicating that a sub-optimal response to the high oxygen stress of culture could be responsible for the accumulation of DNA damage foci and activation of the p53 pathway in AE cultures. Inhibition of p53 by shRNA led to increased resistance of AE cells to gamma irradiation but with continued high levels of DNA damage as shown by phosphorylated H2A.X. Interestingly, the POU4f1 transcription factor, a modifier of the p53 response pathway, has been found to be overexpressed in t(8;21) patient samples. In our system, AE cells that ectopically overexpressed POU4f1 following retroviral transduction showed decreased levels of p53 compared to vector-transduced AE cells. Inhibition of the p53 pathway in pre-leukemic clones expressing AML1-ETO could lead to a proliferative or survival advantage during periods of genotoxic stress. The impairment of DNA repair pathways and the accumulation of DNA damage in AML1-ETO-expressing cells may promote an elevated mutation rate and increase the chances of acquiring subsequent genetic alterations.

scholarly journals Molecular cloning and chromosomal localization of DNA sequences associated with a human DNA repair gene.

1985 ◽  
Vol 5 (2) ◽  
pp. 398-405 ◽  
Author(s):  
J S Rubin ◽  
V R Prideaux ◽  
H F Willard ◽  
A M Dulhanty ◽  
G F Whitmore ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Chromosomal Localization ◽  
Excision Repair ◽  
Repair Process ◽  
Human Cells ◽  
Gene Products ◽  
Repair Gene ◽  
Human Dna ◽  
Dna Repair Gene

The genes and gene products involved in the mammalian DNA repair processes have yet to be identified. Toward this end we made use of a number of DNA repair-proficient transformants that were generated after transfection of DNA from repair-proficient human cells into a mutant hamster line that is defective in the initial incision step of the excision repair process. In this report, biochemical evidence is presented that demonstrates that these transformants are repair proficient. In addition, we describe the molecular identification and cloning of unique DNA sequences closely associated with the transfected human DNA repair gene and demonstrate the presence of homologous DNA sequences in human cells and in the repair-proficient DNA transformants. The chromosomal location of these sequences was determined by using a panel of rodent-human somatic cell hybrids. Both unique DNA sequences were found to be on human chromosome 19.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase

1993 ◽  
Vol 13 (9) ◽  
pp. 5829-5842
Author(s):  
P Zheng ◽  
D S Fay ◽  
J Burton ◽  
H Xiao ◽  
J L Pinkham ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Synthesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Low Frequency ◽  
S Phase ◽  
Upstream Region ◽  
Specific Gene

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

scholarly journals The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

1997 ◽  
Vol 17 (5) ◽  
pp. 2381-2390 ◽  
Author(s):  
A E Parker ◽  
R K Clyne ◽  
A M Carr ◽  
T J Kelly
Keyword(s):  
Dna Repair ◽  
Dna Synthesis ◽  
Schizosaccharomyces Pombe ◽  
Excision Repair ◽  
Protein A ◽  
Simian Virus 40 ◽  
Replication Protein A ◽  
S Phase ◽  
Replication Protein

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

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 Systematic analysis of mutational spectra associated with DNA repair deficiency in C. elegans

2020 ◽  
Author(s):  
B Meier ◽  
NV Volkova ◽  
Y Hong ◽  
S Bertolini ◽  
V González-Huici ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Structural Variants ◽  
Systematic Analysis ◽  
C Elegans ◽  
Mutational Spectra ◽  
Base Substitutions ◽  
Repair Pathways ◽  
Complex Structural

AbstractGenome integrity is particularly important in germ cells to faithfully preserve genetic information across generations. As yet little is known about the contribution of various DNA repair pathways to prevent mutagenesis. Using the C. elegans model we analyse mutational spectra that arise in wild-type and 61 DNA repair and DNA damage response mutants cultivated over multiple generations. Overall, 44% of lines show >2-fold increased mutagenesis with a broad spectrum of mutational outcomes including changes in single or multiple types of base substitutions induced by defects in base excision or nucleotide excision repair, or elevated levels of 50-400 bp deletions in translesion polymerase mutants rev-3(pol ζ) and polh-1(pol η). Mutational signatures associated with defective homologous recombination fall into two classes: 1) mutants lacking brc-1/BRCA1 or rad-51/RAD51 paralogs show elevated base substitutions, indels and structural variants, while 2) deficiency for MUS-81/MUS81 and SLX-1/SLX1 nucleases, and HIM-6/BLM, HELQ-1/HELQ and RTEL-1/RTEL1 helicases primarily cause structural variants. Genome-wide investigation of mutagenesis patterns identified elevated rates of tandem duplications often associated with inverted repeats in helq-1 mutants, and a unique pattern of ‘translocation’ events involving homeologous sequences in rip-1 paralog mutants. atm-1/ATM DNA damage checkpoint mutants harboured complex structural variants enriched in subtelomeric regions, and chromosome end-to-end fusions. Finally, while inactivation of the p53-like gene cep-1 did not affect mutagenesis, combined brc-1 cep-1 deficiency displayed increased, locally clustered mutagenesis. In summary, we provide a global view of how DNA repair pathways prevent germ cell mutagenesis.

Immune checkpoint inhibitor sensitivity of DNA repair deficient tumors

Immunotherapy ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 1205-1213
Author(s):  
Pauline Rochefort ◽  
Françoise Desseigne ◽  
Valérie Bonadona ◽  
Sophie Dussart ◽  
Clélia Coutzac ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Immune Checkpoint ◽  
Genome Stability ◽  
Immune Checkpoint Inhibitor ◽  
Checkpoint Inhibitor ◽  
Excision Repair ◽  
Checkpoint Inhibitors ◽  
Repair Deficiency ◽  
Dna Repair Deficiency

Faithful DNA replication is necessary to maintain genome stability and implicates a complex network with several pathways depending on DNA damage type: homologous repair, nonhomologous end joining, base excision repair, nucleotide excision repair and mismatch repair. Alteration in components of DNA repair machinery led to DNA damage accumulation and potentially carcinogenesis. Preclinical data suggest sensitivity to immune checkpoint inhibitors in tumors with DNA repair deficiency. Here, we review clinical studies that explored the use of immune checkpoint inhibitor in patient harboring tumor with DNA repair deficiency.

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

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadezda V. Volkova ◽  
Bettina Meier ◽  
Víctor González-Huici ◽  
Simone Bertolini ◽  
Santiago Gonzalez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Point Mutations ◽  
Mutational Signatures ◽  
Dna Damage And Repair ◽  
C Elegans ◽  
Dna Repair Deficiency ◽  
Dna Alterations ◽  
Repair Pathways

AbstractCells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.

Mutations in CREBBP and EP300 genes affect DNA repair of oxidative damage in Rubinstein-Taybi syndrome cells

2019 ◽  
Vol 41 (3) ◽  
pp. 257-266
Author(s):  
Ilaria Dutto ◽  
Claudia Scalera ◽  
Micol Tillhon ◽  
Giulio Ticli ◽  
Gianluca Passaniti ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Genome Stability ◽  
Oxidative Dna Damage ◽  
Excision Repair ◽  
Control Cell ◽  
Nuclear Antigen ◽  
Autosomal Dominant Disorder ◽  
Cell Extracts ◽  
Increased Risk

Abstract Rubinstein-Taybi syndrome (RSTS) is an autosomal-dominant disorder characterized by intellectual disability, skeletal abnormalities, growth deficiency and an increased risk of tumors. RSTS is predominantly caused by mutations in CREBBP or EP300 genes encoding for CBP and p300 proteins, two lysine acetyl-transferases (KAT) playing a key role in transcription, cell proliferation and DNA repair. However, the efficiency of these processes in RSTS cells is still largely unknown. Here, we have investigated whether pathways involved in the maintenance of genome stability are affected in lymphoblastoid cell lines (LCLs) obtained from RSTS patients with mutations in CREBBP or in EP300 genes. We report that RSTS LCLs with mutations affecting CBP or p300 protein levels or KAT activity, are more sensitive to oxidative DNA damage and exhibit defective base excision repair (BER). We have found reduced OGG1 DNA glycosylase activity in RSTS compared to control cell extracts, and concomitant lower OGG1 acetylation levels, thereby impairing the initiation of the BER process. In addition, we report reduced acetylation of other BER factors, such as DNA polymerase β and Proliferating Cell Nuclear Antigen (PCNA), together with acetylation of histone H3. We also show that complementation of CBP or p300 partially reversed RSTS cell sensitivity to DNA damage. These results disclose a mechanism of defective DNA repair as a source of genome instability in RSTS cells.

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