scholarly journals Mismatch repair systems might facilitate the chromosomal recombination induced by N-nitrosodimethylamine, but not by N-nitrosodiethylamine, in Drosophila

Mutagenesis ◽  
2020 ◽  
Vol 35 (2) ◽  
pp. 197-206
Author(s):  
Tomoe Negishi ◽  
Kenji Yamada ◽  
Keiko Miyamoto ◽  
Emiko Mori ◽  
Kentaro Taira ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Dna Lesions ◽  
Spot Test ◽  
Dna Double Strand Breaks ◽  
Spontaneous Mutation Rate ◽  
Futile Cycle ◽  
Strand Breaks ◽  
Chromosomal Recombination

Abstract Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(−/−)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/−)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(−/−) flies than in spel1(+/−) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(−/−) flies than in spel1(+/−) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.

scholarly journals Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A

2020 ◽  
Vol 11 ◽  
Author(s):  
Sohail Ahmad ◽  
Qihong Huang ◽  
Jinfeng Ni ◽  
Yuanxi Xiao ◽  
Yunfeng Yang ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Repair Pathway ◽  
Wild Type ◽  
Spontaneous Mutation Rate ◽  
Hyperthermophilic Archaeon ◽  
Cleavage Activity ◽  
Sulfolobus Islandicus ◽  
Mismatch Repair Pathway

EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn2+ was used as the co-factor instead of Mg2+, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10−3) than that of the wild type (4.6 × 10−6). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

scholarly journals The Oxidized Deoxynucleoside Triphosphate Pool Is a Significant Contributor to Genetic Instability in Mismatch Repair-Deficient Cells

2004 ◽  
Vol 24 (1) ◽  
pp. 465-474 ◽  
Author(s):  
Maria Teresa Russo ◽  
Monica Francesca Blasi ◽  
Federica Chiera ◽  
Paola Fortini ◽  
Paolo Degan ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutation Rate ◽  
Genetic Instability ◽  
Mammalian Cells ◽  
Spontaneous Mutation ◽  
Cancer Cell Line ◽  
Spontaneous Mutation Rate ◽  
Mutator Phenotype ◽  
Dntp Pool ◽  
High Level

ABSTRACT Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2 −/− mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of −G and −A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

scholarly journals Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Robert P Fuchs ◽  
Asako Isogawa ◽  
Joao A Paulo ◽  
Kazumitsu Onizuka ◽  
Tatsuro Takahashi ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Futile Cycle ◽  
Repair Synthesis ◽  
Strand Breaks ◽  
Independent Mechanism ◽  
Xenopus Egg ◽  
Cycle Dependent ◽  
Mmr Proteins

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

Verbascoside is not genotoxic in the ST and HB crosses of the Drosophila wing spot test, and its constituent, caffeic acid, decreases the spontaneous mutation rate in the ST cross

2012 ◽  
Vol 50 (3-4) ◽  
pp. 1082-1090 ◽  
Author(s):  
Luis Felipe Santos-Cruz ◽  
José Guillermo Ávila-Acevedo ◽  
Diego Ortega-Capitaine ◽  
Jesús Clemente Ojeda-Duplancher ◽  
Juana Laura Perdigón-Moya ◽  
...  
Keyword(s):  
Caffeic Acid ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Spot Test ◽  
Spontaneous Mutation Rate ◽  
Drosophila Wing

scholarly journals Crosstalk between repair pathways elicits Double-Strand Breaks in alkylated DNA: implications for the action of temozolomide

2020 ◽  
Author(s):  
Robert P. Fuchs ◽  
Asako Isogawa ◽  
Joao A. Paulo ◽  
Kazumitsu Onizuka ◽  
Tatsuro Takahashi ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Futile Cycle ◽  
Repair Synthesis ◽  
Strand Breaks ◽  
Egg Extracts ◽  
Established Cell ◽  
Cycle Dependent ◽  
Xenopus Egg Extracts

AbstractTemozolomide, a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-established cell cycle dependent mode of action.

scholarly journals Inactivation of DNA Mismatch Repair by Increased Expression of Yeast MLH1

2001 ◽  
Vol 21 (3) ◽  
pp. 940-951 ◽  
Author(s):  
Polina V. Shcherbakova ◽  
Mark C. Hall ◽  
Marc S. Lewis ◽  
Samuel E. Bennett ◽  
Karla J. Martin ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Mutation Rate ◽  
Analytical Ultracentrifugation ◽  
Dna Mismatch Repair ◽  
Genome Instability ◽  
Spontaneous Mutation ◽  
Moderate Increase ◽  
Spontaneous Mutation Rate ◽  
Dna Mismatch ◽  
Mutator Effect

ABSTRACT Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177–3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)+ mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3′→5′ exonuclease activity of replicative DNA polymerases δ and ɛ but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, orPMS1. This suggests that overexpression ofMLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K d of 3.14 μM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressingMLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

scholarly journals DNA-RNA hybrids at DSBs interfere with repair by homologous recombination

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pedro Ortega ◽  
Jose Antonio Mérida-Cerro ◽  
Ana G Rondón ◽  
Belén Gómez-González ◽  
Andrés Aguilera
Keyword(s):  
Homologous Recombination ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Positive Role ◽  
Strand Breaks ◽  
Endonucleolytic Cleavage ◽  
Chromosomal Recombination

DNA double strand breaks (DSBs) are the most harmful DNA lesions and their repair is crucial for cell viability and genome integrity. The readout of DSB repair may depend on whether DSBs occur at transcribed versus non-transcribed regions. Some studies have postulated that DNA-RNA hybrids form at DSBs to promote recombinational repair, but others have challenged this notion. To directly assess whether hybrids formed at DSBs promote or interfere with recombinational repair we have used plasmid and chromosomal-based systems for the analysis of DSB-induced recombination in Saccharomyces cerevisiae. We show that, as expected, DNA-RNA hybrid formation is stimulated at DSBs. In addition, mutations that promote DNA-RNA hybrid accumulation, such as hpr1∆ and rnh1∆ rnh201∆, cause high levels of plasmid loss when DNA breaks are induced at sites that are transcribed. Importantly, we show that high levels or unresolved DNA-RNA hybrids at the breaks interfere with their repair by homologous recombination. This interference is observed for both plasmid and chromosomal recombination and is independent of whether the DSB is generated by endonucleolytic cleavage or by DNA replication. These data support a model in which DNA-RNA hybrids form fortuitously at DNA breaks during transcription, and need to be removed to allow recombinational repair, rather than playing a positive role.

A Mutation in a Saccharomyces cerevisiae Gene (RAD3) Required for Nucleotide Excision Repair and Transcription Increases the Efficiency of Mismatch Correction

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 459-466 ◽  
Author(s):  
Yingying Yang ◽  
Anthony L Johnson ◽  
Leland H Johnston ◽  
Wolfram Siede ◽  
Errol C Friedberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Excision Repair ◽  
Spontaneous Mutation ◽  
Surprising Result ◽  
Spontaneous Mutation Rate ◽  
Mismatch Correction ◽  
Nucleotide Excision ◽  
Repair Genes ◽  
Type Strains

Abstract RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMSl, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase δ was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

scholarly journals Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy

2021 ◽  
Vol 22 (14) ◽  
pp. 7638
Author(s):  
Yvonne Lorat ◽  
Judith Reindl ◽  
Anna Isermann ◽  
Christian Rübe ◽  
Anna A. Friedl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dna Damage ◽  
Spatial Clustering ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Stimulated Emission ◽  
Nuclear Chromatin ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Background: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. Material and Methods: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). Results: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. Conclusion: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

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