Unusual Structural Features of Hydantoin Lesions Translate into Efficient Recognition by <i>Escherichia coli</i> Fpg

Krishnamurthy, Nirmala; Muller, James G.; Burrows, Cynthia J.; David, Sheila S.

doi:10.1021/bi602459v

Cited by 28 publications

(57 citation statements)

References 72 publications

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“…S4) suggested that although MmuNeil3Δ324 is bifunctional (with rates of 0.119AE 0.016 min −1 for single-stranded Sp1 and 0.053 AE 0.002 min −1 for double-stranded Sp1 substrates), its lyase activity is not coupled to its glycosylase activity, since the slow lyase reaction of MmuNeil3Δ324 does not limit the enzyme turnover of the glycosylase reaction. MmuNeil3Δ324 has a similar catalytic turnover rate for the glycosylase reaction (3.2 min −1 , Table 1) on double-stranded Sp substrate as does E. coli Nei (1.6-13.0 min −1 (22)(23)(24)) and Fpg (6.0-34.2 min −1 (22)(23)(24)) proteins, although it is much slower than NEIL1 (177 min −1 (25)) on the same substrate. However, MmuNeil3Δ324 exhibits a much higher binding affinity to a double-stranded Sp substrate (K m ¼ 0.753 nM, Table 1), compared to EcoFpg (K m ¼ 25 nM (24)) and EcoNei (K m ¼ 30 nM (24)).…”

Section: Resultsmentioning

confidence: 97%

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

Liu

Bandaru

Bond

et al. 2010

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

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To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Δ324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.base excision repair | endonuclease VIII Like 3 | 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) | Spiroiminodihydantoin D NA glycosylases play an important role in maintaining genomic integrity by recognizing various nonhelix distorting base lesions created by ionizing radiation, alkylating, or oxidizing agents, which are often cytotoxic or mutagenic (1). DNA glycosylases cleave the N-glycosylic bond and release the base lesion from the sugar backbone, resulting in an apurinic or apyrimidinic (AP) site (2). Besides glycosylase activity, some of these enzymes also have a lyase activity to process the AP site. In this way, DNA glycosylases initiate the Base Excision Repair (BER) † pathway and create the substrates for further processing by a series of BER enzymes including phosphodiesterases, AP endonucleases, DNA polymerases, and DNA ligases to complete lesion repair (3).DNA glycosylases that recognize oxidized bases can be divided into two families based on structure and sequence homology (4, 5), the Nth family, whose members are widely distributed in bacteria, archaea, and eukaryotes (5), and the Fpg/Nei family, which is more sparsely distributed across phyla. Fpg/Nei family members are characterized by a signature helix-two turns-helix (H2TH) motif and a zinc finger (or "zincless finger") motif for DNA binding; they also have a conserved N terminus harboring a proline residue (P2) important for catalysis (6). In Escherichia coli, formamidopyrimidine DNA glycosylase (EcoFpg) mainly recognizes oxidized purines (7), and endonuclease VIII (EcoNei) mainly recognizes oxidized pyrimidines and adenin...

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Section: Resultsmentioning

confidence: 97%

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

Liu

Bandaru

Bond

et al. 2010

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

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176

249

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“…EcFpg utilizes an N-terminal proline to catalyze base removal and β,δ-elimination reactions to yield a strand break. This work led to a follow-up paper with greater details about the repair process in a 30-mer duplex leading to many key observations [188]. Diastereomeric mixtures of each hydantoin were studied opposite all four canonical nucleotides to determine whether Sp or Gh were removed by EcFpg as efficiently as the signature substrate OG:C. When Sp or Gh were base paired with A they were operated on by EcFpg more efficiently than an OG:C base pair, and when the hydantoins were opposite C, T, or G they were nearly as good substrates as OG:C. One key point in comparisons between the 18-mer and 30-mer duplex studies is that the duplex length is critical to capturing a true snapshot of the removal kinetics for highly disruptive lesions like the hydantoins in dsDNA.…”

Section: The Hydantoins Are Substrates For Ecfpg Ecnei and Neil1mentioning

confidence: 99%

Formation and processing of DNA damage substrates for the hNEIL enzymes

Fleming

Burrows

2017

Free Radical Biology and Medicine

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Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed.

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“…The hydantoin lesions, Gh and Sp have been shown to be substrates for the E. coli enzymes Fpg (24,39) and Nei (40), and the murine enzymes NEIL1 and NEIL2 (38). In order to elucidate 1 Abbreviations: BER, base excision repair; bp, base pair; EDTA, ethylenediaminetetraacetic acid; Fpg/MutM; bacterial formamidopyrimidine glycosylase; Gh, guanidinohydantoin; hNEIL1, human Nei-like glycosylase 1; Nei; endonuclease VIII; NTH1, endonuclease III homologue 1; OG, 7,8-dihydro-8-oxoguanine; 5-OHC, 5-hydroxycytosine; PAGE, polyacrylamide gel electrophoresis; ROS, reactive oxygen species, Sp, spiroiminodihydantoin; Tg, thymine glycol.…”

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confidence: 99%

Superior Removal of Hydantoin Lesions Relative to Other Oxidized Bases by the Human DNA Glycosylase hNEIL1

et al. 2008

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The DNA glycosylase hNEIL1 initiates the base excision repair (BER) of a diverse array of lesions, including ring-opened purines and saturated pyrimidines. Of these, the hydantoin lesions, guanidinohydantoin (Gh) and the two diastereomers of spiroiminodihydantoin (Sp1 and Sp2) have garnered much recent attention due to their unusual structures, high mutagenic potential and detection in cells. In order to provide insight into the role of repair, the excision efficiency by hNEIL1 of these hydantoin lesions relative to other known substrates was determined. Most notably, quantitative examination of the substrate specificity with hNEIL1 revealed that the hydantoin lesions are excised much more efficiently (> 100-fold faster) than the reported standard substrates thymine glycol (Tg) and 5-hydroxycytosine (5-OHC). Importantly, the glycosylase and β,δ-lyase reactions are tightly coupled such that the rate of the lyase activity does not influence the observed substrate specificity. The activity of hNEIL1 is also influenced by the base pair partner of the lesion, with both Gh and Sp removal being more efficient when paired with T, G or C than when paired with A. Notably, the most efficient removal is observed with the Gh or Sp paired in the unlikely physiological context with T; indeed, this may be a consequence of the unstable nature of base pairs with T. However, the facile removal via BER in promutagenic base pairs that are reasonably formed after replication (such as Gh:G) may be a factor that modulates the mutagenic profile of these lesions. In addition, hNEIL1 excises Sp1 faster than Sp2 indicating the enzyme can discriminate between the two diastereomers. This is the first time that a BER glycosylase has been shown to be able to preferentially excise one diastereomer of Sp. This may be a consequence of the architecture of the active site of hNEIL1 and the structural uniqueness of the Sp lesion. These results indicate that the hydantoin lesions are the best substrates identified thus far for hNEIL1, and suggest that repair of these lesions may be a critical function of the hNEIL1 enzyme in vivo.Cells experiencing oxidative stress have an overabundance of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals (1,2). ROS are present in cells as byproducts of endogenous metabolic reactions or as a result of external sources such as ionizing radiation. The reactions mediated by ROS can lead to various types of DNA damage including strand breaks, DNA-protein cross-links, abasic sites, and base lesions, which are potentially detrimental to cells (3)(4)(5)(6). Oxidative DNA damage is mitigated by a variety of DNA repair pathways (7-9). The importance of repairing DNA damage has been highlighted by the correlation between defects in DNA repair pathways and cancer (7,(10)(11)(12). ¶ This work was supported by a grant from the National Cancer Institute of the National Institutes of Health (CA90689). Although a variety of guanine oxidation products have been identified (13), most st...

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Unusual Structural Features of Hydantoin Lesions Translate into Efficient Recognition by Escherichia coli Fpg

Cited by 28 publications

References 72 publications

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

Formation and processing of DNA damage substrates for the hNEIL enzymes

Superior Removal of Hydantoin Lesions Relative to Other Oxidized Bases by the Human DNA Glycosylase hNEIL1

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