Formation and processing of DNA damage substrates for the hNEIL enzymes

Abstract

Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed.

Keywords: 5-carboxamido-5-formamido-2-iminohydantoin; 5-guanidinohydantoin; 8-oxo-7,8-dihydroguanine; G-quadruplex; NEIL DNA glycosylase; Spiroiminodihydantoin.

Figure 1

Mechanisms for ROS formation, redox potentials for nucleosides, ROS, and reductants. (A) Reaction schemes for generation of ROS. (B) Plot of the redox potentials for nucleosides, reductants (Asc = ascorbate, NAC = N-acetylcysteine, GSH = glutathione), and various ROS.

Figure 2

Context dependency of major G oxidation pathways and products. The contexts studied in DNA include nucleoside, ssDNA, dsDNA, and G4 structures and the products shown are those appearing during oxidations conducted in the presence of physiological amounts of reductant. In several cases, OG is an initial product that undergoes subsequent oxidation to Sp as indicated by straight arrows, or Sp is directly formed from oxidation of G bypassing OG as indicated by no arrow between OG and Sp.

Figure 3

BER mechanism and relative reaction rates for NEIL1 removal of DNA lesions. (A) Outline of the BER pathway (dRP = 2′-deoxyribosephosphate). (B) Comparison of edited NEIL1 reaction rates vs. DNA lesions relative to Tg:A (arrow). The inset is for unedited NEIL1 reaction rates relative to Tg:G (arrow). (C) Edited NEIL1 reaction rates toward the diastereomers of Sp and Gh vs. their base pairing partner. (D) Context-dependent reaction rates for edited NEIL1 relative to the ssDNA context (arrow).

Figure 4

NEIL initiated repair in the VEGF G4 requires the 5^th G track to allow a structural transition to a repair-competent fold.

Figure 5

Context and substrate preference for MmuNeil3 and cellular properties of the NEIL glycosylases. (A) Efficiency of removal for various substrates by MmuNeil3 and MmuNeil3Δ324 in dsDNA contexts. (B) Efficiency of removal for various substrates by MmuNeil3 and MmuNeil3Δ324 in ssDNA contexts. The graphs in parts A and B are adapted from the original publication on this work [204]. (C) Table to compare the NEILs on the basis of their substrate preference, reaction context preference, protein interaction partners, and cell cycle of maximal expression. The graphs in part A are adapted from the original publication [204].

Scheme 1

Overview of G oxidation products and the extent to which each was oxidized leading to their formation. The products inside the dashed boxes are the main focus of this review because they are the best substrates for the hNEIL glycosylases (2Ih, Sp, or Gh) or a key precursor to these products (OG).

Scheme 2

HO^•-mediated oxidation of the G heterocycle. The dominate pathways are shown with bold reaction arrows. The compounds in boxes are the focus of this review because they have been identified as the best substrates for the hNEIL glycosylases in the BER pathway.

Scheme 3

Proposed one-electron oxidation mechanisms of the G heterocycle. The key products repaired by hNEIL1 resulting from this mechanism are identified by the dashed boxes. RSH = cellular reductant such as glutathione.

Scheme 4

Proposed mechanism for oxidation of the G heterocycle by ¹O₂. The key products repaired by the hNEILs are within the dashed boxes.