DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

Yuri N. Vorobjev,Maria V. Lukina,Alexander V. Popov,Olga S. Fedorova,Vladimir V. Koval,Dmitry O. Zharkov

doi:10.1074/jbc.m113.487322

Abstract

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.

Highlights

Oxoguanine-DNA glycosylase (OGG1) removes highly mutagenic 8-oxoguanine from DNA
A long-standing problem in the field of DNA repair is how DNA repairs enzymes, DNA glycosylases in particular, and discriminates their target-damaged nucleotides from the normal ones, which are present in a great excess yet, with very rare exceptions, are all but refractory to the enzymes’ action
The helical axis of DNA is kinked at the point of the lesion (ϳ70° in OGG1-DNA complex 1EBM), and the damaged nucleotide is rotated from its position inside the double helix into the enzyme’s active site

Summary

Background

Oxoguanine-DNA glycosylase (OGG1) removes highly mutagenic 8-oxoguanine from DNA. Results: OGG1 mutations C253I and C253L occlude the active site and distort the OGG1-DNA precatalytic complex but retain some activity. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. Pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate ␤-elimination. To understand the importance of the interactions within the base-binding pocket for different reactions catalyzed by OGG1 on different substrates, we have constructed site-directed mutants replacing the Cys-253 residue with either leucine or isoleucine (Fig. 1A). The active site of OGG1 appears to be flexible enough to retain some residual repair activity

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION