Critical sites of DNA backbone integrity for damaged base removal by formamidopyrimidine–DNA glycosylase

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 t...