UC Davis

The genomic information of all living organisms is susceptible to alterations through chemical modifications in DNA occurring from both environmental and cellular sources. To prevent the detrimental effects of DNA damage, DNA repair pathways were acquired and evolved to target and repair certain modifications in DNA. Guanine Oxidation (GO) repair pathway is one of the several pathways that uses DNA glycosylases to target DNA damage resulting from oxidation of guanine. The adenine glycosylase MutY is one of the key players in this pathway that removes the undamaged adenine (A) base across an oxidation product of guanine (G), 8-oxo- guanine (OG).

The focus of this work is to understand the mechanistic and structural details of how MutY locates and differentiates its target mispair from other similar base pairs and excises the misincorporated adenine. The significance of MutY in maintaining the integrity of DNA is highlighted by its relevance to inherited colorectal cancer syndrome known as MUTYH-associated polyposis (MAP). Previous studies revealed the importance of the C-terminal domain (CTD) of the protein in OG recognition and the roles of the residues in the active site of N-terminal domain (NTD) in adenine excision mechanism. The goal of my thesis work is to elucidate roles of residues involved in OG recognition and adenine excision mechanisms by using enzymology, nucleic acid chemistry and X-ray crystallography.

This work reveals the role of a CTD loop comprising Phe, Ser and His residues, referred as FSH loop, that presumably involved in OG recognition provided by structural implications. Our structural and biochemical results showed the significance of FSH loop within the CTD, in OG versus G recognition. Indeed, the replacement of more than one residue in the FSH loop results in loss of specificity for OG. Furthermore, the replacement of serine in the loop affected the overall activity of MutY, albeit showing similar specificity as the wild-type enzyme. We believe the reduced activity with FAH is due to the involvement of serine residue in a hydrogen bond network around OG. The residues in this hydrogen bond network may all work in conjunction to ensure faithful OG recognition and adenine placement in the active site.

The OG lesion is known to pair with adenine in syn conformation in duplex DNA, however OG is found in anti conformation in the crystal structures of MutY bound to DNA. The hydrogen bond network around OG in the anti conformation involves contacts with serine of FSH loop and two NTD Gln and Thr residues. The Gln and Thr residues from NTD intercalates into the space left from extruded adenine. We explored the roles of these intercalation residues by mutational analysis and structure activity relationship (SAR) studies with a series of previously studied OG analogs. The kinetic and binding results indicated Gln and Thr residues may have a role in OGanti recognition and in proper placement of adenine in the active site for removal.

The recent structural and biochemical studies from David and Horvath Laboratories led to a proposed double replacement mechanism for MutY. In this mechanism, a highly conserved catalytic Asp residue is proposed to form a covalent intermediate. We replaced the Asn residue that is the hydrogen partner of catalytic Asp to study the additional features of the adenine excision mechanism. The structural and kinetics results of Asn146Ser variant indicated that the loss of hydrogen bonding to Asp resulted in significantly impaired activity and an increase in the pKa of Asp. Next, we obtained a crystal structure of N146S in complex with a an alternative substrate OG:Purine (P). To our surprise, we captured the OG:P substrate before N-glycosidic bond hydrolysis occurred in the active site. In addition, a calcium ion coordinated to the base and catalytic Asp was found in the space previously occupied by Asn side chain that is likely to be responsible for the inhibition of the base removal. The further crystallization trials with extended incubation time or removal of calcium afforded the first structures of Asn146Ser variant in complex with enzyme-catalyzed β-anomer AP site formation consistent with retaining mechanism. The combination of the crystal structures forms a structural movie of base excision mechanism by N146S variant that provides further insight into the MutY adenine excision mechanism.

In the final chapter, I describe a computational study on MutY structures in complex with either a substrate or transition state analog to identify the hot spots of the enzyme for ligand binding. These models and computational results showed an Arg residue may either open or close the end of the active site channel. We further investigated the role of Arg by replacing it with a Gly or Trp residue to either open or close the channel, respectively. Our biochemical findings suggested Arg may be indirectly affecting the adenine excision and this region may be targeted as an allosteric site for drug discovery.

Taken all together, this work sheds light into the roles of residues involved in OG recognition, adenine positioning and excision in the active site. Furthermore, we identified the FSH loop and adenine exit channel as allosteric sites that can affect the adenine excision activity indirectly. The development of small molecule inhibitors targeting these regions may provide the basis for novel cancer chemotherapeutics.