Abstract
The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.
Highlights
The internal components of aerobic cells are under chronic bombardment by electrophilic agents known collectively as reactive oxygen species [1]
Cells defend themselves against nucleobase lesions such as oxoG through the operation of the evolutionarily conserved base excision DNA repair pathway (6 – 8), the key components of which are DNA glycosylases, lesion-specific enzymes that scan the genome for aberrant nucleobases and catalyze their excision [9, 10]
DNA glycosylases belonging to each of the four known structural superfamilies of DNA glycosylases, two of which are represented by MutM and Ogg1, contain in their active site pocket conserved residues that serve critical roles in catalysis plus variable residues that are responsible for conferring the unique lesion recognition preferences of individual enzymes
Summary
The internal components of aerobic cells are under chronic bombardment by electrophilic agents known collectively as reactive oxygen species [1]. A close interaction between these two active site residues has been observed in the x-ray structure of a hOGG1 LRC containing Lys-249 but having another catalytically essential residue, Asp-268, mutated to Asn (Fig. 1A and Ref. 26).
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