5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

Abstract

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.

Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA

“…Our work describes the first crystal structure of any Nei in complex with a damaged base. Several structures of Fpg in complex with a lesion (8-oxoG, ring-opened formamidopyrimidine, and 5-hydroxy-5-methylhydantoin) have been reported (15,16,42,56), and these structures were compared with the MvNei1 complexes to gauge how lesions are recognized. Among the Fpg structures, the BstFpgE3Q⅐8oxoG:C complex was chosen as a reference because the E3Q variant was employed in both BstFpg and MvNei1 structures (15).…”

Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

“…Assays were performed in standard experimental conditions (21,22) except that all incubation mixtures contained 8% final concentration of dimethyl sulfoxide (DMSO) required for solubilizing the nucleobases (G, 8-oxoG, FapyG, X and 2TX, Supplementary Figure S1a). After electrophoresis, gels were exposed to autoradiography; scanned using STORM-Imager and quantified using ImageQuant software.…”

The Fpg/Nei Family of DNA Glycosylases

“…Over the past decade, there has been a significant increase in the number of crystal structures of Fpg/Nei glycosylases (54-69). The advent of techniques such as reductive cross-linking using sodium borohydride has played an essential role in trapping stable protein-DNA complexes for the purposes of crystallization and to elucidate the mechanism and role of these intricate enzymes ((54-56,64-66,70,71) and for reviews see (70,71)).…”