DNA Deformation-Coupled Recognition of 8-Oxoguanine: Conformational Kinetic Gating in Human DNA Glycosylase

Abstract

8-Oxoguanine (8-oxoG), a mutagenic DNA lesion generated under oxidative stress, differs from its precursor guanine by only two substitutions (O8 andH7). Human 8-oxoguanine glycosylase 1 (OGG1) can locate and remove 8-oxoG through extrusion and excision. To date, it remains unclear how OGG1 efficiently distinguishes 8-oxoG from a large excess of undamaged DNA bases. We recently showed that formamidopyrimidine–DNA glycosylase (Fpg), a bacterial functional analog of OGG1, can selectively facilitate eversion of oxoG by stabilizing several intermediate states, and it is intriguing whether OGG1 also employs a similar mechanism in lesion recognition. Here, we use molecular dynamics simulations to explore the mechanism by which OGG1 discriminates between 8-oxoG and guanine along the base eversion pathway. The MD results suggest an important role for kinking of the DNA by the glycosylase, which positions DNA phosphates in a way that assists lesion recognition during base eversion. The computational predictions were validated through experimental enzyme assays on phosphorothioate substrate analogs. Our simulations suggest that OGG1 distinguishes between 8-oxoG and G using their chemical dissimilarities not only at the active site, but also at earlier stages during base eversion, and this mechanism is at least partially conserved in Fpg despite lack of structural homology. The similarity also suggests that lesion recognition through multiple gating steps may be a common theme in DNA repair. Our results provide new insight into how enzymes can exploit kinetics and DNA conformational changes to probe the chemical modifications present in DNA lesions.

Reading and Misreading 8-oxoguanine, a Paradigmatic Ambiguous Nucleobase

“…An extended series of structures of Fpg [102][103][104][105][127][128][129][130][131] and OGG1 [110,120,121,123,[132][133][134][135][136][137][138], together with molecular dynamic simulation of conformational transition between these intermediates [139][140][141][142], and stopped-flow studies [115,122,140,[143][144][145][146][147][148][149][150] produced a multistep oxoG recognition model that turned out to be surprisingly similar for these two enzymes despite their structural dissimilarity. Both Fpg and OGG1 kink DNA by 40 • -50 • and insert an aromatic wedge (Phe or Tyr) into the base stack, causing the sampled base pair to buckle.…”

A Fast Implementation of the Nudged Elastic Band Method in AMBER

“…This necessitates statistical analysis of multiple independent simulations which result in different pathways-which once again illustrates the importance of fast simulation techniques. 28,29 Implementation GPU programming is inevitably susceptible to the latency of the data transfer between the device and the host memory which hinders the performance of the GPU-accelerated code. In the PNEB routines, specifically, the coordinate exchange between the replicas at each step of the simulation can aggravate the performance.…”

“…different aspects of this transition to better understand the base eversion pathways and the preferred binding conformation. 28,29,32,38,39 The two endpoints correspond to the intrahelical and the extrahelical conformations in the base eversion pathway. Figure 8 illustrates the endpoint structures of the region involved in the transition plus the glycosidic angle versus the eversion distance change of this transition.…”

“…This eversion distance has been reported previously as a reaction coordinate for nucleic acid base eversion. 29,40 The red points are the result of the simulations performed with nebfreq equal to 1. The light blue points are the result of the simulations performed with nebfreq equal to 5 in all the stages of the simulation but the first and the final, at which the value of nebfreq was set to 2.…”

“…As previously illustrated in the work of Bergonzo et al16 and Li et al,29 a Langevin thermostat with a high collision frequency is required to control the temperature when performing NEB simulations. High values of collision frequency may not be appropriate for normal MD simulations, but it is recommended for NEB simulations.…”