Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ72–47protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that overexpress MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.

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Effect of DNA Glycosylases OGG1 and Neil1 on Oxidized G-Rich Motif in the KRAS Promoter

International Journal of Molecular Sciences ◽

10.3390/ijms22031137 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1137

Author(s):

Annalisa Ferino ◽

Luigi E. Xodo

Keyword(s):

Oxidative Stress ◽

Base Excision Repair ◽

Excision Repair ◽

Start Site ◽

Dna Glycosylases ◽

Repair Pathway ◽

Transcription Start ◽

G Quadruplex ◽

Base Excision ◽

Regulatory Functions

The promoter of the Kirsten ras (KRAS) proto-oncogene contains, upstream of the transcription start site, a quadruplex-forming motif called 32R with regulatory functions. As guanine under oxidative stress can be oxidized to 8-oxoguanine (8OG), we investigated the capacity of glycosylases 8-oxoguanine glycosylase (OGG1) and endonuclease VIII-like 1 (Neil1) to excise 8OG from 32R, either in duplex or G-quadruplex (G4) conformation. We found that OGG1 efficiently excised 8OG from oxidized 32R in duplex but not in G4 conformation. By contrast, glycosylase Neil1 showed more activity on the G4 than the duplex conformation. We also found that the excising activity of Neil1 on folded 32R depended on G4 topology. Our data suggest that Neil1, besides being involved in base excision repair pathway (BER), could play a role on KRAS transcription.

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The Role of Base Excision Repair in the Sensitivity and Resistance to Temozolomide-Mediated Cell Death

Cancer Research ◽

10.1158/0008-5472.can-05-0715 ◽

2005 ◽

Vol 65 (14) ◽

pp. 6394-6400 ◽

Cited By ~ 133

Author(s):

Ram N. Trivedi ◽

Karen H. Almeida ◽

Jamie L. Fornsaglio ◽

Sandra Schamus ◽

Robert W. Sobol

Keyword(s):

Cell Death ◽

Base Excision Repair ◽

Excision Repair ◽

Base Excision

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Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

Frontiers in Microbiology ◽

10.3389/fmicb.2021.608839 ◽

2021 ◽

Vol 12 ◽

Author(s):

Almaz Nigatu Tesfahun ◽

Marina Alexeeva ◽

Miglė Tomkuvienė ◽

Aysha Arshad ◽

Prashanna Guragain ◽

...

Keyword(s):

Escherichia Coli ◽

Excision Repair ◽

Dna Lesions ◽

Base Pairs ◽

E Coli ◽

Thymine Glycol ◽

Base Excision ◽

The Cost ◽

Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

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Bending of DNA duplexes with mutation motifs

DNA Research ◽

10.1093/dnares/dsz013 ◽

2019 ◽

Vol 26 (4) ◽

pp. 341-352

Author(s):

Michal Růžička ◽

Přemysl Souček ◽

Petr Kulhánek ◽

Lenka Radová ◽

Lenka Fajkusová ◽

...

Keyword(s):

De Novo ◽

Excision Repair ◽

Dna Duplexes ◽

De Novo Mutations ◽

Base Pairs ◽

Inherited Disorders ◽

Cpg Dinucleotides ◽

Human Genes ◽

Base Excision ◽

Dynamics Simulations

Abstract Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.

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Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide

Biochemical Journal ◽

10.1042/bj2350531 ◽

1986 ◽

Vol 235 (2) ◽

pp. 531-536 ◽

Cited By ~ 76

Author(s):

M Dizdaroglu ◽

E Holwitt ◽

M P Hagan ◽

W F Blakely

Keyword(s):

Dna Repair ◽

Formic Acid ◽

Excision Repair ◽

Dna Lesions ◽

Gas Chromatography Mass Spectrometry ◽

Dna Glycosylases ◽

Thymine Glycol ◽

Repair Enzymes ◽

Base Excision

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

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Role of nucleotide- and base-excision repair in genotoxin-induced neuronal cell death

DNA Repair ◽

10.1016/j.dnarep.2004.02.005 ◽

2004 ◽

Vol 3 (6) ◽

pp. 617-627 ◽

Cited By ~ 22

Author(s):

G.E Kisby ◽

H Lesselroth ◽

A Olivas ◽

L Samson ◽

B Gold ◽

...

Keyword(s):

Cell Death ◽

Base Excision Repair ◽

Excision Repair ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Base Excision

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A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

DNA Repair ◽

10.1016/j.dnarep.2007.01.009 ◽

2007 ◽

Vol 6 (6) ◽

pp. 695-711 ◽

Cited By ~ 275

Author(s):

Karen H. Almeida ◽

Robert W. Sobol

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Protein Complexes ◽

Post Translational Modification ◽

Unified View ◽

Dependent Protein ◽

Base Excision

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Evidence for the Involvement of Nucleotide Excision Repair in the Removal of Abasic Sites in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3522-3528.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3522-3528 ◽

Cited By ~ 56

Author(s):

Carlos A. Torres-Ramos ◽

Robert E. Johnson ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Repair Process ◽

Dna Glycosylases ◽

Abasic Sites ◽

Skin Cancers ◽

Nucleotide Excision ◽

Progressive Neurodegeneration ◽

Ap Sites ◽

Base Excision

ABSTRACT In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment ∼25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Δ orapn1Δ apn2Δ strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

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