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 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 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.

EXPRESSION IN DIPLOIDS OF THE MUTATOR PHENOTYPE OF SOME MUTATOR MUTANTS OF SACCHAROMYCES CEREVISIAE

1980 ◽  
Vol 22 (1) ◽  
pp. 51-59 ◽  
Author(s):  
D. P. Morrison ◽  
Siew-Keen Quah ◽  
P. J. Hastings
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutation Rate ◽  
Mating Type ◽  
Spontaneous Mutation ◽  
Spontaneous Mutation Rate ◽  
Mutator Phenotype ◽  
Recombination Repair

The mutator mutants of yeast, mut3-1, mut3-2 and mut4-1, are much less clearly expressed in diploids than in haploids, but the mutator phenotypes of mut3-1 and mut4-1 are restored when the diploids are made homozygous for the a mating type. An allele of RAD51, mut5-1, is expressed in a/a diploids to the same extent as in a/a diploids. Since a mutation of RAD51 causes loss of the a/α recombination repair after λ-irradiation, it is concluded that these two a/α effects are expressions of the same mechanism and that, therefore, mut3, mut4 and mut5 all owe their high spontaneous mutation rate to changes in the repair of spontaneous lesions in DNA. It is noted that, in the absence of a/α repair, the spontaneous mutation rate per cell in a diploid is the same as, rather than twice, that in a haploid.

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 Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants.

Genetics ◽  
1994 ◽  
Vol 138 (3) ◽  
pp. 553-564 ◽  
Author(s):  
M E Santos ◽  
J W Drake
Keyword(s):  
Dna Polymerase ◽  
Mutation Rate ◽  
Excision Repair ◽  
Bacteriophage T4 ◽  
Spontaneous Mutation ◽  
Resolving Power ◽  
Mutation Rates ◽  
Spontaneous Mutation Rate ◽  
Dntp Pool ◽  
Host Fidelity

Abstract Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.

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 Mechanisms of Dinucleotide Repeat Instability in Escherichia coli

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 533-542
Author(s):  
Marc Bichara ◽  
Isabelle Pinet ◽  
Sylvie Schumacher ◽  
Robert P P Fuchs
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Genetic Instability ◽  
Positional Cloning ◽  
Dinucleotide Repeats ◽  
Disease Pathogenesis ◽  
Complementary Sequence ◽  
Phylogenetic Studies ◽  
High Level

Abstract The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies. The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability. In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli. We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability. We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)-containing repeats. For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures.

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