Credit: Courtesy of A. Banerjee

Oxidative damage can create 8-oxoguanine (oxoG) lesions on G bases of DNA. If left unrepaired, oxoG is read as a T by the replication machinery, subsequently resulting in a G-C to T-A conversion after a second round of DNA replication. Human 8-oxoguanine DNA glycosylase I (hOGG1) recognizes oxoG-C lesions and initiates base excision repair to remove them. The change from G to oxoG is subtle (addition of O to C8 and protonation of N7), so understanding how hOGG1 discriminates between the two is of interest, particularly because the normal base is so abundant in DNA. A previous structure of oxoG-containing DNA bound to a catalytically inactive form of the enzyme showed that oxoG swings out from the double helix to bind hOGG1 at the recognition pocket (protein and DNA in green, oxoG in red ball-and-stick), leaving its partner cytosine (magenta) 'estranged.' To see whether hOGG1 interacts with nondamaged DNA in a different manner, which may account for the discrimination, Banerjee et al. (Nature 434 612–618, 2005) examined complexes of hOGG1 bound to duplex DNA containing a normal G-C base pair.

Crystallization of such a complex is difficult because repair proteins have an inherently weak affinity for DNA that lacks the cognate lesions they recognize. To overcome this problem, the authors engineered a disulfide bond between hOGG1 and the DNA outside the oxoG-binding pocket, between the cytosine of the G-C pair and a newly introduced cysteine on hOGG1. The strategy allowed them to examine the complex crystallographically.

The structure of the complex (protein and DNA in gold, G in blue ball-and-stick) shares an overall similarity with the oxoG-hOGG1 structure, but the G is extruded from the helix and lies at a new site 5 Å outside of the lesion-recognition pocket. Storage of the G-complex over ten days revealed no discernable base excision activity, in contrast to a similar covalent complex containing the oxoG lesion. The authors used free energy simulations to compare binding contributions at the oxoG-binding pocket and at the exosite and found a ∼105-fold preference for oxoG over G at the lesion-binding pocket. Additional calculations revealed changes in a single hydrogen bond and a local dipole moment between oxoG and G that allows stable active site interactions with oxoG but not with G if it were to enter the pocket. These results likely explain why G is not found in the lesion-recognition pocket in the hOGG1-G structure.

Verdine and colleagues suggest that the G-complex represents a late intermediate in the base extrusion for oxoG. When combined with the structure of free hOGG1, the structures of the G- and oxoG-complexes outline the conformational changes that occur as the base is inserted into the lesion-recognition pocket. Although the analysis provides a means for extrahelical oxo-G versus G base discrimination, it is unknown whether hOGG1 can discriminate between bases that are not extruded from the double helix. Nonetheless, hOGG1 seems to have a very efficient strategy in place to prevent cleavage of normal, lesion-free DNA.