Trapping and structural elucidation of a very advanced intermediate in the lesion-extrusion pathway of hOGG1

Here we present the first structure of a very advanced intermediate in the lesion-extrusion pathway of a DNA glycosylase, human 8-oxoguanine DNA glycosylase (hOGG1), and a substrate DNA containing a mutagenic lesion, 8-oxoguanine (oxoG). The structure was obtained by irradiation and flash-freezing of a disulfide-cross-linked (DXLed) complex of hOgg1 bound to DNA containing a novel photocaged derivative of oxoG. The X-ray structure reveals that upon irradiation, the oxoG lesion has transited from the exosite to the active site pocket, but has not undergone cleavage by the enzyme. Furthermore, all but one of the specificity-determining interactions between the lesion and the enzyme are unformed in the flashed complex (FC), because active site functionality and elements of the DNA backbone are mispositioned. This structure thus provides a first glimpse into the structure of a very late-stage intermediate in the lesion-extrusion pathway-the latest observed to date for any glycosylase-in which the oxoG has undergone insertion into the enzyme active site following photodeprotection, but the enzyme and DNA have not yet completed the slower process of adjusting to the presence of the lesion in the active site.