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

Abstract

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...

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

“…3). Under conditions of multiple turnover, NEIL1 exhibits biphasic "burst" kinetics with Sp-containing substrates providing a means to accurately determine the active site fraction (14). Both enzyme forms exhibit similar active fractions indicating that the difference in amino acid at this position does not globally alter protein folding or stability needed for activity.…”

“…This enzyme is capable of removing a wide array of modified DNA bases including thymine glycol (Tg), 5-hydroxycytosine (5-OHC), 5-hydroxyuracil (5-OHU), dihydrothymine (DHT), dihydrouracil (DHU), the formamidopyridines (FapyG and FapyA), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) (Fig. 1) (12)(13)(14)(15). Oxidized base lesions arise in DNA at rates of thousands per day as a result of endogenous metabolic activity as well as from oxidative stress induced by inflammation, radiation, or toxic agents (16).…”

“…Oxidized base lesions arise in DNA at rates of thousands per day as a result of endogenous metabolic activity as well as from oxidative stress induced by inflammation, radiation, or toxic agents (16). The most thoroughly examined oxidized base is 8-oxo-7,8-dihydroguanine (OG) which is one of the primary substrates for the human OG glycosylase (hOGG1) but is not efficiently processed by NEIL1 (12,14,16). The best substrates documented for NEIL1 are the hydantoin lesions (Gh and Sp) that form from further oxidation of OG or from reactions with potent oxidants such as singlet oxygen (12,14).…”

“…The most thoroughly examined oxidized base is 8-oxo-7,8-dihydroguanine (OG) which is one of the primary substrates for the human OG glycosylase (hOGG1) but is not efficiently processed by NEIL1 (12,14,16). The best substrates documented for NEIL1 are the hydantoin lesions (Gh and Sp) that form from further oxidation of OG or from reactions with potent oxidants such as singlet oxygen (12,14). These lesions have garnered much attention due to their extremely high mutagenic potential in cells which is significantly greater than OG (16).…”

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

“…Oligodeoxyribonucleotides with Tg, 8-oxoG, or 5-OHU as well as all of the non-lesion-containing oligodeoxyribonucleotides were purchased from Midland Certified Reagent Co. (Midland, TX). Gh-and Sp-containing substrates were synthesized as described previously (40). The Tel-Sp oligodeoxyribonucleotides used here were a mixture of Sp1 and Sp2 due to the inability to resolve these diastereomers under the purification method utilized.…”