Cynthia J. Burrows

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

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.

Alkene aziridination and epoxidation catalyzed by chiral metal salen complexes

Abstract New chiral metal salen complexes substituted in the 3 position with bulky silyl groups (TMS, TBDMS) have been synthesized. Although some of these complexes catalyzed the epoxidation of unfunctionalized olefins in moderate to high ees, they catalyzed the aziridination of cis-β-methylstyrene without any asymmetric induction.

Superior removal of hydantoin lesions relative to other oxidized bases by the human DNA glycosylase hNEIL1.

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 beta,delta-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.

Mechanistic studies of alkene epoxidation catalyzed by nickel(II) cyclam complexes. Oxygen-18 labeling and substituent effects.

The oxidations of cyclohexene and various aryl-substituted alkenes are catalyzed by the cyclam (1,4,8,11-tetraazacyclotetradecane) complex of Ni(NO/sub 3/)/sub 2/ with iodosylbenzene as terminal oxidant. Epoxides are the major products; however, small amounts of ring-opened products, over-oxidation to ketones or aldehydes, and allylic oxidation of cyclohexene are also observed. E olefins are more reactive than the corresponding Z olefins in contrast to the results of iron porphyrin catalysis, and kinetic studies of para-substituted styrenes indicate that the reaction is facilitated by electron-donating substituents. Labeling studies with PhI/sup 18/O confirm that the epoxide oxygen is derived from PhIO while allylic oxidation and over-oxidation products involve both PhIO and exogenous sources of oxygen. A pericyclic mechanism for the formation of PhCHO is proposed along with the intermediacy of a high-valent nickel-oxo complex as the active oxidant. These results are discussed in light related transition-metal/PhIO oxidation mechanisms.

Formation of DNA adducts using nickel(II) complexes of redox-active ligands: a comparison of salen and peptide complexes

Abstract A series of water-soluble Ni(salen) complexes have been synthesized and studied under oxidative conditions with DNA. Experiments involving gel electrophoretic analysis, substituent effect studies, and mass spectral characterization point to the formation of covalent adducts of the salen ligand with guanine nucleobases. The mechanism of this novel reactivity of transition metal complexes with nucleic acids is discussed in comparison with nickel(II) complexes of peptides and other ligands.

An exploration of mechanisms for the transformation of 8-oxoguanine to guanidinohydantoin and spiroiminodihydantoin by density functional theory.

The potential energy surface for formation of 2-amino-5-hydroxy-7,9-dihydropurine-6,8-dione (5-OH-OG), guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from 8-oxoguanine (8-oxoG) has been mapped out using B3LYP density functional theory, the aug-cc-pVTZ and 6-31+G(d,p) basis sets and the IEF-polarizable continuum model (PCM) solvation model. Three pathways for formation of 5-OH-OG from 8-oxoG were evaluated: (A) stepwise loss of two electrons and two protons to form the quinonoid intermediate 2-amino-7,9-dihydro-purine-6,8-dione (8-oxoG(ox)) followed by hydration; (B) stepwise loss of two electrons and one proton and net addition of hydroxide, in which the key step is nucleophilic addition to the 8-oxoG radical cation; and (C) stepwise loss of one electron and one proton and addition of hydroxyl radical to the 8-oxoG radical cation. The data suggest that all three pathways are energetically feasible mechanisms for the formation of 5-OH-OG, however, Pathway A may be kinetically favored over Pathway B. Although lower in energy, Pathway C may be of limited biological significance since it depends on the local concentration of hydroxyl radical. Pathways for hydrolysis and decarboxylation of 5-OH-OG to form Gh via either a carboxylic acid or substituted carbamic acid intermediate have been evaluated with the result that cleavage of the N1-C6 bond is clearly favored over that of the C5-C6 bond. Formation of Sp from 5-OH-OG via stepwise proton transfer and acyl migration or ring opening followed by proton transfer and ring closure have also been explored and suggest that deprotonation of the hydroxyl group facilitates a 1,2 acyl shift. Results of the calculations are consistent with experimental studies showing dependence of the Gh/Sp product ratio on pH. Under neutral and basic conditions, the data predict that formation of Sp is kinetically favored over the pathways for formation of Gh. Under acidic conditions, Gh is predicted to be the kinetically favored product.

Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na+ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li+, Na+ or K+) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement.

Nanopore Detection of 8-Oxo-7,8-dihydro-2′-deoxyguanosine in Immobilized Single-Stranded DNA via Adduct Formation to the DNA Damage Site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.

Alkene Epoxidation Using Ni(II) Complexes of Chiral Cyclams

Abstract A series of optically active Ni(II)-cyclam complexes are active catalysts for the PhIO oxidation of alkenes; labeling and stereochemical studies suggest important mechanistic differences from heme catalysts.

Chemical modification of siRNA bases to probe and enhance RNA interference

Considerable attention has focused on the use of alternatives to the native ribose and phosphate backbone of small interfering RNAs for therapeutic applications of the RNA interference pathway. In this synopsis, we highlight the less common chemical modifications, namely, those of the RNA nucleobases. Base modifications have the potential to lend insight into the mechanism of gene silencing and to lead to novel methods to overcome off-target effects that arise due to deleterious protein binding or mis-targeting of mRNA.