The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of the base excision repair (BER) pathway initiated by the bifunctional DNA glycosylase NEIL1. In this work, we reported that the excision kinetics of the single Sp lesions site-specifically embedded in the covalently closed circular DNA plasmids (contour length 2686 base pairs) by NEIL1 are biphasic under single-turnover conditions ([NEIL1] ≫ [SpDNApl]) in contrast with monophasic excision kinetics of the same lesions embedded in147-mer Sp-modified DNA duplexes. Under conditions of a large excess of plasmid DNA base pairs over NEIL1 molecules, the kinetics of excision of Sp lesions are biphasic in nature, exhibiting an initial burst phase, followed by a slower rate of formation of excision products The burst phase is associated with NEIL1–DNA plasmid complexes, while the slow kinetic phase is attributed to the dissociation of non-specific NEIL1–DNA complexes. The amplitude of the burst phase is limited because of the competing non-specific binding of NEIL1 to unmodified DNA sequences flanking the lesion. A numerical analysis of the incision kinetics yielded a value of φ ≍ 0.03 for the fraction of NEIL1 encounters with plasmid molecules that result in the excision of the Sp lesion, and a characteristic dissociation time of non-specific NEIL1–DNA complexes (τ-ns ≍ 8 s). The estimated average DNA translocation distance of NEIL1 is ∼80 base pairs. This estimate suggests that facilitated diffusion enhances the probability that NEIL1 can locate its substrate embedded in an excess of unmodified plasmid DNA nucleotides by a factor of ∼10.

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