Structure-activity relationships guided studies for OG:A lesion recognition and repair by the base excision glycosylase MutY

The presence of reactive oxygen species (ROS) in cells can lead to the oxidation of the DNA base guanine (G) to 8-oxo-7,8-dihydroguanine (OG) (Fig 1a). This oxidative product has the ability to aberrantly code like a thymine, leading to the conversion of G:C base pairs to OG:A mis-pairs and then further to T:A base pairs (Fig 1b). Although the OG:A pair thus formed is structurally almost identical to an undamaged T:A base pair (Fig 1c), the base excision repair (BER) glycosylase, MutY is proficient at locating these lesions within the genome and catalyzing the removal of the mis-paired adenine. This step triggers a cascade of reactions by downstream repair enzymes that ultimately result in the reinstatement of the original G:C base pair. The critical role of the MutY glycosylase in maintaining genomic integrity is underscored by the link between deficiencies in OG:A repair by the human homolog, MUTYH, and a colorectal cancer syndrome known as MUTYH associated polyposis (MAP).
We seek to understand the structural basis by which MutY identifies the OG:A lesion and distinguishes it from a normal, undamaged T:A through structure-activity relationships (SAR). Through the incorporation of substrate analogs of OG and A into DNA duplexes, we can evaluate the effects of modified steric, electronic and base pairing properties on in vitro parameters such as enzyme-substrate binding and kinetics of base cleavage, as well as on overall repair in a bacterial cell assay. These studies help us gauge the importance of the different structural features of the bases on the recognition and repair process of MutY. Furthermore, applying this insight to the crystal structure of the substrate-bound enzyme, we can potentially identify the regions and residues on MutY that are responsible for lesion recognition. Based on these analyses, we investigate the effect of mutations to these residues by purifying MutY variants and utilizing the aforementioned in vitro and cellular assays to characterize them. Taken together, these studies will help us understand the complete mechanism of MutY’s search and damage recognition process within the genome.