Effect of the Oxidized Guanosine Lesions Spiroiminodihydantoin and Guanidinohydantoin on Proofreading by Escherichia coli DNA Polymerase I (Klenow Fragment) in Different Sequence Contexts

Abstract

Oxidative damage to DNA by endogenous and exogenous reactive oxygen species has been directly linked to cancer, aging, and a variety of neurological disorders. The potential mutagenicity of the primary guanine oxidation product 8-oxo-7,8-dihydroguanine (Og) has been studied intensively, and much information is available about its miscoding potential in vitro and in vivo. Recently, a variety of DNA lesions have been identified as oxidation products of both guanine and 8-oxoguanine, among them spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). To address questions concerning the mutagenic potential of these secondary products of guanine oxidation, the effect of the lesions on proofreading by DNA polymerase was studied in vitro using the Klenow fragment of Escherichia coli polymerase I (Kf exo+). For the first time, k(cat)/K(m) values were obtained for proofreading of the X:N mismatches (X = Og, Gh, or Sp; N = A, G, or C). Proofreading studies of the terminal mismatches demonstrated the significance of the sequence context flanking the lesion on the 3' side. In addition, a sequence dependence was observed for Gh based on the identity of the base on the 5' side of the lesion providing evidence for a primer slippage mode if N was complementary to the 5' base. Internal mismatches were recognized by Kf exo+ resulting in the excision of the correct base pairs flanking mismatches from the 5' side. The absence of a sequence effect for the Gh- and Sp-containing duplexes can be attributed to the severe destabilization of the lesion-containing duplexes that promotes interaction with the exonuclease domain of the Klenow fragment.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

“…However, a recent study showed that in E. coli, the mutagenicity of 8-oxoG is greater than that of FapyG (32). It is also possible that the reduced mutagenicity we observed in the triple mutant strain is partially due to the further oxidation product of 8-oxoG, Sp, which gives rise to a combination of Gua → Thy and Gua → Cyt transversion mutations (33,34) and can be recognized by MmuNeil3. Sp has been shown to accumulate in Nei deficient E. coli cells exposed to chromate which requires Nei for its repair (35).…”

Determination of the stability constants and oxidation susceptibility of nickel(II) complexes with 2′-deoxyguanosine 5′-triphosphate and l-histidine

“…Such products were, indeed, obtained before under various conditions from dG and identified as oxaluric acid, spiroiminodihydantoin, and guanidinohydantoin [59][60][61]. Some of them were then proven to be mutagenic [61]. An issue of their generation in our complex systems requires further investigations.…”

“…The low yield of 8-oxo-dGTP, compared with the 30% loss of the original dGTP at the same time, but not through any detectable dephosphorylation, clearly indicates that 8-oxo-dGTP was further oxidized to other guanine products, which evaded UV detection in our HPLC analyses. Such products were, indeed, obtained before under various conditions from dG and identified as oxaluric acid, spiroiminodihydantoin, and guanidinohydantoin [59][60][61]. Some of them were then proven to be mutagenic [61].…”

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

“…Further work is necessary to confirm the R and S designations of the monomer and oligomers. Of note, Gh also exists as a mixture of diastereomers, but these interconvert on a time scale that is too rapid to allow isolation and independent study (53).Thermal denaturation experiments have found that the thermodynamic stability of Spcontaining oligonucleotides is significantly reduced (ΔΔG 37 ~ 6 kcal/mol) compared to OG or G counterparts (25,26,54). Molecular dynamics simulations also indicated that the Sp nucleotide would be destabilizing to the DNA duplex and that the R isomer of Sp would be less destabilizing than the S isomer within a DNA duplex (55).…”