NEIL1 Binding to DNA Containing 2′‐Fluorothymidine Glycol Stereoisomers and the Effect of Editing

Abstract

Thymine glycol (Tg), one of the oxidized bases formed in DNA by reactive oxygen species, is repaired by the DNA glycosylases such as NEIL1, NTH1 and Endo III. In our recent studies, we showed that NEIL1’s catalytic efficiency and lesion specificity are regulated by an RNA editing adenosine deamination reaction. In this study, we synthesized oligodeoxynucleotides containing 2′-fluorothymidine glycol with either ribo or arabino configuration and investigated the binding of these modified DNAs with the unedited and edited forms of human NEIL1 along with E. coli Endo III. For the two forms of hNEIL1, binding affinities to FTg-containing DNA were similar indicating the editing effect is more subtle than to simply alter substrate affinity. While the NEIL1 binding to FTg-containing DNAs was largely insensitive to C5 and 2′ stereochemistry, a preference was observed for the FTg-G pair over the FTg-A pair. In addition, we found that optimal binding is observed with Endo III and duplex DNA with riboFTg (5S) paired with dG. The modified DNAs reported here will provide useful tools for further characterizing the interaction between DNA repair glycosylases and thymine glycol containing DNA.

Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

“…AP site binding competition assay was performed with 20 pM of an AP site containing double stranded oligonucleotide, preincubated with 50 µg/ml BSA (Control) or 500 nM TDG in binding buffer for 10 min at room temperature before adding 500 µg/ml BSA (Control), 250 nM NEIL1 or 750 nM NEIL2 for another 10 min. PAGE was done as described previously58 but with 10% native polyacrylamide gels. Phosphorimaging was done on a Typhoon FLA 9500 (GE Healthcare) and quantification using ImageQuant TL software (GE Healthcare).…”

The emerging role of RNA editing in plasticity

“…In addition, mRNAs encoding the DNA repair enzyme NEIL1 can be edited at a lysine codon to produce an arginine (Li et al, 2009). This edit redirects NEIL1 from removing thymine glycol in dsDNA to the repair of guanidinohydantoin lesions (Onizuka et al, 2012;Yeo et al, 2010). It is intriguing to think that editing might influence DNA repair in a manner that is consistent with environmental insults.…”

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

“…Furthermore, introduction of 2′-deoxy-2′-fluoroarabinofuranosyl thymine ( FdT ) residues at the cross-link site shunted repair of the cross-link to the incision-dependent (Fanconi anemia) pathway shown in Figure 1. This result strongly implicated glycosylase activity in repair of the psoralen cross-link because the electron-withdrawing effect of the 2′-fluoro substituent in FdT (Figure 2C) inhibits the action of BER glycosylases through destabilization of the oxocarbenium ion-like transition state of these enzymatic reactions [81, 82, 106, 107]. …”