Inactivation of DNA Mismatch Repair by Increased Expression of Yeast MLH1

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.

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

Frontiers in Microbiology ◽

10.3389/fmicb.2020.607431 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.465-474.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 465-474 ◽

Cited By ~ 100

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.

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

Mutagenesis ◽

10.1093/mutage/geaa008 ◽

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.

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Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.407-415.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 407-415

Author(s):

T A Prolla ◽

D M Christie ◽

R M Liskay

Keyword(s):

Mismatch Repair ◽

Dna Mismatch Repair ◽

Genetic Recombination ◽

Sequence Similarity ◽

Spontaneous Mutation ◽

Protein Product ◽

Mlh1 Gene ◽

Dna Mismatch ◽

Postmeiotic Segregation ◽

Simple Sequence

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.

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A genome-wide screen identifies genes that suppress the accumulation of spontaneous mutations in young and aged yeast cells

10.1101/492587 ◽

2018 ◽

Author(s):

Daniele Novarina ◽

Georges Janssens ◽

Koen Bokern ◽

Tim Schut ◽

Noor van Oerle ◽

...

Keyword(s):

Mutation Rate ◽

Genome Stability ◽

Genome Instability ◽

Spontaneous Mutation ◽

Yeast Cells ◽

Spontaneous Mutations ◽

Genome Maintenance ◽

Spontaneous Mutation Rate ◽

Genome Wide ◽

A Genome

To ensure proper transmission of genetic information, cells need to preserve and faithfully replicate their genome, and failure to do so leads to genome instability, a hallmark of both cancer and aging. Defects in genes involved in guarding genome stability cause several human progeroid syndromes, and an age-dependent accumulation of mutations has been observed in different organisms, from yeast to mammals. However, it is unclear if the spontaneous mutation rate changes during aging, and if specific pathways are important for genome maintenance in old cells. We developed a high-throughput replica-pinning approach to screen for genes important to suppress the accumulation of spontaneous mutations during yeast replicative aging. We found 13 known mutation suppression genes, and 31 genes that had no previous link to spontaneous mutagenesis, and all acted independently of age. Importantly, we identified PEX19, encoding an evolutionarily conserved peroxisome biogenesis factor, as an age-specific mutation suppression gene. While wild-type and pex19Δ young cells have similar spontaneous mutation rates, aged cells lacking PEX19 display an elevated mutation rate. This finding suggests that functional peroxisomes are important to preserve genome integrity specifically in old cells, possibly due to their role in reactive oxygen species metabolism.

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