Recognition and repair of oxidatively generated DNA lesions in plasmid DNA by a facilitated diffusion mechanism

Abstract

The oxidatively generated genotoxic spiroiminodihydantoin (Sp) lesions are well-known substrates of 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 to 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 in amplitude 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.

Distinct Mechanisms of Target Search by Endonuclease VIII-like DNA Glycosylases

“…Together with this study, there are now four H2TH DNA glycosylases characterized to any extent with respect to the search mechanism: Fpg, Nei, NEIL1 and NEIL2 [ 13 , 14 , 20 , 21 , 23 , 24 , 46 ]. Fpg seems to be the most clear-cut case.…”

“…Much to our surprise, there was no noticeable decrease in the P cc values over the entire range of intersite distances ( Figure 3 ). In a recent report on the lesion search in plasmid substrates by human NEIL1, the estimated mean translocation distance was approximately 80 bp [ 46 ]. Given that NEIL1 here and in [ 46 ] showed a typical salt dependence profile, it is possible that NEIL1 is indeed processive and a decrease in P cc would be observed at distances > 80 bp, which are not easily achieved in an oligonucleotide-based system.…”

“…In a recent report on the lesion search in plasmid substrates by human NEIL1, the estimated mean translocation distance was approximately 80 bp [ 46 ]. Given that NEIL1 here and in [ 46 ] showed a typical salt dependence profile, it is possible that NEIL1 is indeed processive and a decrease in P cc would be observed at distances > 80 bp, which are not easily achieved in an oligonucleotide-based system. On the other hand, P cc of Nei apparently did not depend on the intersite distance, which, together with weak salt dependence, is indicative of the low processivity of target search by this enzyme (see the Discussion for factors affecting the bulk processivity observed in double-cleavage experiments).…”

“…Many proteins that recognize specific sequences, chemical modifications or structural elements in DNA, despite possessing quite different structural folds, share a common mechanism of target search, namely facilitated one-dimensional diffusion along the DNA contour. In particular, such a mechanism was demonstrated for a number of DNA glycosylases, the enzymes that recognize damaged nucleobases and initiate base excision DNA repair [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 46 ]. Here, we show that in a group of structurally related DNA glycosylases, all belonging to the same H2TH structural superfamily, may vary greatly in their ability to carry out processive lesion search, which, therefore, should not be taken for granted as a universal target location mechanism.…”

“…Mechanisms of lesion search by Nei and NEILs remain poorly studied: diffusion constants for Escherichia coli Nei under different conditions were previously obtained using the single-molecule DNA tightrope assay [ 23 , 24 ], and the processivity of human NEIL1 was recently explored using kinetic analysis on a circular plasmid [ 46 ]. Here, using a biochemical processivity assay based on the cleavage of an oligonucleotide with two lesions [ 7 , 47 ], we have investigated the contribution of the facilitated diffusion into the lesion search by E. coli Nei and murine NEIL1 and NEIL2.…”