Differential repair enzyme-substrate selection within dynamic DNA energy landscapes.

Abstract

We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10-12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also 'induce' rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.