Guanine Oxidation Product 5-Carboxamido-5-formamido-2-iminohydantoin Induces Mutations When Bypassed by DNA Polymerases and Is a Substrate for Base Excision Repair

Abstract

Guanine (G) is a target for oxidation by reactive oxygen species in DNA, RNA and the nucleotide pool. Damage to DNA yields products with alternative properties toward DNA processing enzymes compared to the parent nucleotide. A new lesion, 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) bearing a stereocenter in the base, was recently identified from the oxidation of G. DNA polymerase and base excision repair processing of this new lesion has now been evaluated. Single nucleotide insertion opposite (S)-2Ih and (R)-2Ih in the template strand catalyzed by the DNA polymerases Klenow fragment exo−, DPO4, and Hemo KlenTaq demonstrate these lesions to cause point mutations. Specifically, they promote threefold more G•C → C•G transversion mutations than G•C → T•A, and (S)-2Ih was twofold more blocking for polymerase bypass than (R)-2Ih. Both diastereomer lesions were found to be substrates for the DNA glycosylases NEIL1 and Fpg, and poorly excised by endonuclease III (Nth). The activity was independent of the base pair partner. Thermal melting, CD spectroscopy, and density functional theory geometric optimization calculations were conducted to provide insight into these polymerase and DNA glycosylase studies. These results identify that formation of the 2Ih lesions in a cell would be mutagenic in the event that they were not properly repaired.

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

“…Because the action of APE1 on an AP results in increased transcription, the present experiments address how oxidations yielding APs, such as LSD1-generated H 2 O 2 followed by Fenton chemistry, can change the transcriptional output of a gene without requiring OG. However, other G oxidation products from Fenton chemistry, such as the hydantoin products (24,25), are substrates for the NEIL1 and 2 glycosylases and do not require APE1 (28,29) and, therefore, would not impact transcription via the mechanism outlined unless additional factors are involved. Thus, further questions remain concerning how an indiscriminate oxidant like H 2 O 2 liberated by LSD1 or LSD2 would have evolved to write OG at critical locations for gene induction.…”

Formation and processing of DNA damage substrates for the hNEIL enzymes

“…Because OG is always observed in higher concentrations, it is regarded as a good indicator for monitoring cellular oxidative stress. In light of recent in vitro oxidations of G to yield 2Ih in greater levels than OG [62, 154], 2Ih may exist in cells at high levels; however, in cellulo studies to detect 2Ih have yet to be reported…”

“…When 2Ih is in the template strand, dATP or dGTP are inserted opposite the lesion by a polymerase in vitro with a preference for dGTP [154]. Verification of 2Ih causing mutations in cellulo by the REAP assay consistent with the in vitro studies has yet to be conducted.…”

“…The longer duplexes provided a better context for glycosylase binding and catalysis of lesion removal. In a recent study from our laboratory, we found that EcFpg could catalyze excision of each diastereomer of 2Ih opposite A, C, T, or G in a 15-mer duplex with ~50% of the efficiency of removal of Sp from the same duplex [154]. Moreover, the removal efficiency showed little bias for the R or S stereochemistry of 2Ih.…”

“…Such studies were conducted in our laboratory in collaboration with the David laboratory, the Dalhus laboratory, and the Ide laboratory for NEIL1 [154, 192–194]. To date, extensive kinetic analysis for NEIL2 or NEIL3 vs. substrate identity has yet to be reported.…”