Base excision repair of the N-(2-deoxy-d-erythro-pentofuranosyl)-urea lesion by the hNEIL1 glycosylase

Abstract

The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and β deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1′ of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4′ facilitates attack at deoxyribose C1′. The deoxyribose is in the ring-opened configuration with the O4′ oxygen protonated. The electron density of Lys242 suggests the ‘residue 242-in conformation’ associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

Replication Bypass of the N-(2-Deoxy-d-erythro-pentofuranosyl)-urea DNA Lesion by Human DNA Polymerase η

“…The urea-adducted oligodeoxynucleotide obtained from base hydrolysis of thymine glycol (Tg) , containing oligodeoxynucleotides existed as an equilibrium mixture of two species (Figure ). These could be transiently separated by RP-HPLC on a C 18 column at pH 8.0, but subsequently underwent re-equilibration to produce a mixture with the original composition.…”

“…Both were purified by RP-HPLC using a Gemini C 18 250 mm × 10 mm column (Phenomenex, Inc., Torrance, CA). To prepare site-specific urea adducts in this sequence for replication assays, 80 nmol of the Tg-containing oligodeoxynucleotide were hydrolyzed overnight in 0.2 M sodium phosphate (pH 12.0) ,,, at room temperature. Like before, the product 5′- TCAT X ATGACGCTTACGA-3′ ( X = urea lesion) existed as an equilibrium mixture of two species.…”

“…Complexes with Non-hydrolyzable dNTP Analogs. In DNA, urea lesions exist as mixtures of αand β-anomers 7 that, although chromatographically separable, re-equilibrate in solution.…”

“… The initial synthesis of site-specific urea lesions in DNA was accomplished by Teoule and co-workers . In nucleosides , and single-stranded DNA, urea lesions equilibrate between α- and β-anomeric configurations (Scheme ). The urea lesion, ,, like an abasic site (AP site), − lacks its original coding information.…”

Molecular Mechanism of RNA Polymerase II Transcriptional Mutagenesis by the Epimerizable DNA Lesion, Fapy·dG

“…Under some conditions, Fapy·dG is even formed in higher amounts than the widely studied 8-OxodGuo in vitro and in cells. ,– Fapy·dG presents unusual structural properties to a polymerase that stem from the cleavage of the 5-membered imidazole ring of the purine, which increases the number of accessible conformations. The most distinctive structural feature of Fapy·dG is that it is a rare example of a DNA lesion that exists as a mixture of configurational isomers that equilibrate via an imine intermediate (Scheme ), which independently affect genetic expression. – …”