Removal of Hydantoin Products of 8-Oxoguanine Oxidation by the Escherichia coli DNA Repair Enzyme, FPG

Abstract

An intriguing feature of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is that it is highly reactive toward further oxidation. Indeed, OG has been shown to be a "hot spot" for oxidative damage and susceptible to oxidation by a variety of cellular oxidants. Recent work has identified two new DNA lesions, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), resulting from one-electron oxidation of OG. The presence of Gh and Sp lesions in DNA templates has been shown to result in misinsertion of G and A by DNA polymerases, and therefore, both are potentially mutagenic DNA lesions. The base excision repair (BER) glycosylases Fpg and MutY serve to prevent mutations associated with OG in Escherichia coli, and therefore, we have investigated the ability of these two enzymes to process DNA duplex substrates containing the further oxidized OG lesions, Gh and Sp. The Fpg protein, which removes OG and a variety of other oxidized purine base lesions, was found to remove Gh and Sp efficiently opposite all four of the natural DNA bases. The intrinsic rate of damaged base excision by Fpg was measured under single-turnover conditions and was found to be highly dependent upon the identity of the base opposite the OG, Gh, or Sp lesion; as expected, OG is removed more readily from an OG:C- than an OG:A-containing substrate. However, when adenine is paired with Gh or Sp, the rate of removal of these damaged lesions by Fpg was significantly increased relative to the rate of removal of OG from an OG:A mismatch. The adenine glycosylase MutY, which removes misincorporated A residues from OG:A mismatches, is unable to remove A paired with Gh or Sp. Thus, the activity of Fpg on Gh and Sp lesions may dramatically influence their mutagenic potential. This work suggests that, in addition to OG, oxidative products resulting from further oxidation of OG should be considered when evaluating oxidative DNA damage and its associated effects on DNA mutagenesis.

DNA Lesion Recognition by the Bacterial Repair Enzyme MutM

“…Recognition of an Alternative Substrate-In addition to oxoG, MutM repairs a number of alternative substrates, most notably formamidopyrimidine (37-39), 5-hydroxycytosine (40), and the hydantoin lesions (41). We tested the activity of MutM on DNA containing DHU, the product resulting from reduction of the C5ϭC6 double bond in uridine to furnish the corresponding singly bonded CH5-CH6 system.…”

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…We generated a substrate for Fpg by treating λ DNA with methylene blue (MB) plus visible light (24)(25)(26). Treatment of DNA with MB results not only in the formation of 8-oxoG but primarily its further oxidation products such as spiroiminodihydantoin (27,28), which are also recognized by Fpg (29). A substrate containing Tg was generated for Nei and Nth using osmium tetroxide (OsO 4 ) plus heat (30-32) (Materials and Methods).…”

Escherichia coli Apurinic-Apyrimidinic Endonucleases Enhance the Turnover of the Adenine Glycosylase MutY with G:A Substrates

“…The purification of endonuclease IV was modified from Haas et al (62) by replacing dialysis steps with an Amersham Biosciences, Inc. HiPrep 26/10 desalting column. E. coli MutM was purified as described previously (63). Purified Ape1 was a generous gift of Dr. David Wilson and Dr. Jan Erzberger (Lawrence Livermore National Laboratory).…”