Critical Sites of DNA Backbone Integrity for Damaged Base Removal by Formamidopyrimidine–DNA Glycosylase

Abstract

DNA glycosylases, the enzymes that initiate base excision DNA repair, recognize damaged bases through a series of precisely orchestrated movements. Most glycosylases sharply kink the DNA axis at the lesion site and extrude the target base from the DNA double helix into the enzyme's active site. Little attention has been paid so far to the role of the physical continuity of the DNA backbone in allowing the required conformational distortion. Here, we analyze base excision by formamidopyrimidine−DNA glycosylase (Fpg) from substrates keeping all phosphates but containing a nick within three nucleotides of the lesion in either DNA strand. Four phosphoester linkages at the damaged nucleotide and two nucleotides 3′ to it were essential for Fpg activity, while the breakage of the others, even at the same critical phosphates, had no effect or even stimulated the reaction. Reduction of the likelihood of hydrogen bonding at the nicks by using dideoxynucleotides as their 3′-terminal groups was more detrimental for the activity. All phosphoester bonds in the complementary strand were dispensable for base excision, but nicks close to the orphaned nucleotide caused early termination of damaged strand cleavage. Elastic network analysis of Fpg−DNA structures showed that the vibrational motions of the critical phosphates are strongly correlated, in part due to the presence of the protein. Overall, our results suggest that mechanical forces propagating along the DNA backbone play a critical role in the correct conformational distortion of DNA by Fpg and possibly by other target base-everting DNA glycosylases.

Synergistic Antigenotoxic and Antioxidant Action of Gum Arabic and Eugenol in Rat Liver Following Induction of Colorectal Carcinogenesis

“…The alcaline comet assay and the micronucleus assay revealed genotoxicity in all DMH groups. This was confirmed by the modified alkaline comet assay, which is more sensitive and detects specific lesions through the oxidation of nucleotides (Endutkin and Zharkov, 2019).…”

Noncatalytic Domains in DNA Glycosylases

“…This pinching is a necessary step in the damaged nucleoside eversion mechanism required to flip the lesion out of the double helix and into the enzyme's active site [73,75,82]. Except for this Arg, the analysis of intramolecular residue coevolution [83] and protein structure vibrational modes [84] in Fpg shows that the zinc finger is largely uncoupled from the rest of the protein, thus being a domain in the strict sense. Unlike many conventional zinc fingers that recognize specific sequences in DNA and are often present as clustered units in the protein, the H2TH glycosylases' fingers are solitary and contribute a single absolutely conserved Arg residue to the active site (Figure 3A) where it participates in a clamp pinching two phosphates that flank the damaged nucleoside.…”

“…This pinching is a necessary step in the damaged nucleoside eversion mechanism required to flip the lesion out of the double helix and into the enzyme's active site [73,75,82]. Except for this Arg, the analysis of intramolecular residue coevolution [83] and protein structure vibrational modes [84] in Fpg shows that the zinc finger is largely uncoupled from the rest of the protein, thus being a domain in the strict sense.…”