A network of enzymes involved in repair of oxidative DNA damage in <i>Neisseria meningitidis</i>

Nagorska, Krzysztofa; Šilhán, Jan; Li, Yanwen; Pelicic, Vladimir; Freemont, Paul S.; Baldwin, Geoff; Tang, Christoph M.

doi:10.1111/j.1365-2958.2012.07989.x

Cited by 20 publications

(14 citation statements)

References 68 publications

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“…Full-length MutM untagged gene from Neisseria meningitides (strain MC58, UniProt P55044) was expressed and purified as described previously [38]. In brief, protein was expressed in E. coli BL21 Star (DE3) or Roset-taTM(DE3) pLys strains.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…Due to its pathogenic character and restricted habitat, its small genome contains only a handful of DNA repair genes with one Fpg/Nei glycosylase [36,37]. Besides the studying of this important human pathogen, we utilized the limited repertoire of DNA repair enzymes in this bacterium to reveal the network of enzymatic pathways and molecular mechanisms of its DNA repair enzymes [38,39]. Therefore, Neisseria offers us an excellent model of a minimal DNA repair system.…”

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confidence: 99%

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Conformational changes of DNA repair glycosylase MutM triggered by DNA binding

Landová

Šilhán

2020

FEBS Letters

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Bacterial MutM is a DNA repair glycosylase removing DNA damage generated from oxidative stress and, therefore, preventing mutations and genomic instability. MutM belongs to the Fpg/Nei family of prokaryotic enzymes sharing structural and functional similarities with their eukaryotic counterparts, for example, NEIL1–NEIL3. Here, we present two crystal structures of MutM from pathogenic Neisseria meningitidis: a MutM holoenzyme and MutM bound to DNA. The free enzyme exists in an open conformation, while upon binding to DNA, both the enzyme and DNA undergo substantial structural changes and domain rearrangement. Our data show that not only NEI glycosylases but also the MutMs undergo dramatic conformational changes. Moreover, crystallographic data support the previously published observations that MutM enzymes are rather flexible and dynamic molecules.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

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confidence: 99%

Conformational changes of DNA repair glycosylase MutM triggered by DNA binding

Landová

Šilhán

2020

FEBS Letters

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“…All reactions, including metal ion titrations, were performed using oligonucleotide substrates and reaction conditions as previously described (27). Data were fitted to the Michaelis-Menten equation using Grafit 6 (Erithacus software).…”

Section: Methodsmentioning

confidence: 99%

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Lü

Šilhán

MacDonald

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.X-ray crystallography | genome stability | prokaryote A basic (apurinic-apyrimidic, AP) sites within DNA pose a serious threat to all living organisms; if unrepaired, they lead to stalled replication, increased mutations, and an overall loss of genomic integrity. For example human cells defective in AP-site processing are nonviable (1). AP sites occur through either the spontaneous loss of nucleotide bases via chemical breakage of the N-glycosylic bond or as intermediates in the repair of DNA lesions such as oxidatively damaged bases, spontaneously deaminated cytosines, or alkylation of bases (2). It is estimated that 10 4 abasic sites are produced per day per cell in eukaryotes and if left unrepaired will result in significant mutagenic loading on the cell (3, 4). All living organisms have therefore evolved specific enzymes that recognize and repair damaged or missing DNA bases known as the base excision repair (BER) pathway (2).In BER, DNA glycosylases recognize and remove damaged bases to produce abasic sites. The resultant abasic sites are then recognized by AP endonucleases that bind and catalyze a magnesium-dependent 5′ cleavage of the phosphodiester backbone, producing a 3′-OH. Other bifunctional DNA glycosylases cleave the resulting abasic site via an AP lyase mechanism, which leaves a 3′ unsaturated aldehyde (5). In either case, DNA backbone cleavage marks the AP site for subsequent repair by DNA polymerases and ligases. Two main families of AP endonucleases have been identified: the major human AP endonuclease (APEX1) and Escherichia coli homolog ExoIII belong to the ExoIII family of AP endonucleases and are highly conserved (∼51% sequence homology; ∼29% sequence identity). The other is the EndoIV family, named after the E. coli enzyme, and they cleave abasic sites in a Zn 2+ -dependent reaction. Both E. coli and Saccharomyces cerevisiae possess both ExoIII and EndoIV type enzymes, whereas humans have two ExoIII family enzymes.Molecular and biochemical details of abasic DNA recognition and backbone cleavage have mainly focused on the human APEX1 enz...

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“…This result may be attributable to the lack of cytotoxic DNA single-stranded breaks or 3= blocking lesions created when MutM independently or MutY in concert with Nth, Nfo, and XthA removes 8-oxo-dG or -A, respectively, from the DNA upon exposure to H 2 O 2 . Support for this possibility is provided by the increased survival of exonuclease and endonuclease mutants of N. meningitidis or Salmonella enterica serovar Typhimurium under conditions of ROS or RNS stress, respectively, following inactivation of mutM (52,53). Alternatively, the lack of ROS-induced cytotoxic DNA lesions could also be due to effective cleansing of increased 8-oxo-dG from the nucleotide pools by one or multiple MutTs.…”

Section: Resultsmentioning

confidence: 99%

Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

Canfield¹,

Schwingel²,

Foley³

et al. 2012

Journal of Bacteriology

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Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of ␣-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of ␣-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.

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A network of enzymes involved in repair of oxidative DNA damage in Neisseria meningitidis

Cited by 20 publications

References 68 publications

Conformational changes of DNA repair glycosylase MutM triggered by DNA binding

Conformational changes of DNA repair glycosylase MutM triggered by DNA binding

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

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