Abstract
As part of an overall effort to map the energetic landscape of the base excision repair pathway, we report the first thermodynamic characterization of repair enzyme binding to lesion-containing duplexes. Isothermal titration calorimetry (ITC) in conjunction with spectroscopic measurements and protease protection assays have been employed to characterize the binding of
Escherichia coli formamidopyrimidine-glycosylase (Fpg), a bifunctional repair enzyme, to a series of 13-mer DNA duplexes. To resolve energetically the binding and the catalytic events, several of these duplexes are constructed with non-hydrolyzable lesion analogs that mimic the natural 8-oxo-dG substrate and the abasic-like intermediates. Specifically, one of the duplexes contains a central, non-hydrolyzable, tetrahydrofuran (THF) abasic site analog, while another duplex contains a central, carbocyclic substrate analog (carba-8-oxo-dG). ITC-binding studies conducted between 5.0
°C and 15.0
°C reveal that Fpg association with the THF-containing duplex is characterized by binding free energies that are relatively invariant to temperature (Δ
G∼−9.5
kcal
mol
−1), in contrast to both the reaction enthalpy and entropy that are strongly temperature-dependent. Complex formation between Fpg and the THF-containing duplex at 15
°C exhibits an unfavorable association enthalpy
(
ΔH=+7.5
kcal
mol
−1)
that is compensated by a favorable association entropy (
TΔ
S=+17.0
kcal
mol
−1). The entropic nature of the binding interaction, coupled with the large negative heat capacity
(
ΔC
p=−0.67
kcal
deg
−1
mol
−1),
is consistent with Fpg complexation to the THF-containing duplex involving significant burial of non-polar surface areas. By contrast, under the high ionic strength buffer conditions employed herein (200
mM NaCl), no appreciable Fpg affinity for the carba-8-oxo-dG substrate analog is detected. Our results suggest that initial Fpg recognition of a damaged DNA site is predominantly electrostatic in nature, and does not involve large contact interfaces. Subsequent base excision presumably facilitates accommodation of the resulting lesion site into the binding pocket, as the enzyme interaction with the THF-containing duplex is characterized by high affinity and a large negative heat capacity change. Our data are consistent with a pathway in which Fpg glycosylase activity renders the base excision product a preferred ligand relative to the natural substrate, thereby ensuring the fidelity of removing highly reactive and potentially mutagenic abasic-like intermediates through catalytic elimination reactions.