当前位置： SCI文献检索 > JOURNAL OF MOLECULAR BIOLOGY期刊下所有文献 > Energetics of lesion recognition by a DNA repair protein: thermodynamic characterization of formamidopyrimidine-glycosylase (Fpg) interactions with damaged DNA duplexes.

Energetics of lesion recognition by a DNA repair protein: thermodynamic characterization of formamidopyrimidine-glycosylase (Fpg) interactions with damaged DNA duplexes.

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 degrees C and 15.0 degrees C reveal that Fpg association with the THF-containing duplex is characterized by binding free energies that are relatively invariant to temperature (deltaG approximately -9.5 kcalmol(-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 degrees C exhibits an unfavorable association enthalpy (deltaH=+7.5 kcalmol(-1)) that is compensated by a favorable association entropy (TdeltaS=+17.0 kcalmol(-1)). The entropic nature of the binding interaction, coupled with the large negative heat capacity (deltaC(p)=-0.67 kcaldeg(-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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Minetti CA,Remeta DP,Zharkov DO,Plum GE,Johnson F,Grollman AP,Breslauer KJ

doi

10.1016/s0022-2836(03)00365-6

keywords:

subject

Has Abstract

pub_date

2003-05-16 00:00:00

pages

1047-60

issue

eissn

0022-2836

issn

1089-8638

pii

S0022283603003656

journal_volume

328

pub_type

杂志文章

在线工具

Energetics of lesion recognition by a DNA repair protein: thermodynamic characterization of formamidopyrimidine-glycosylase (Fpg) interactions with damaged DNA duplexes.