STRUCTURAL BASIS FOR THE SUBSTRATE SPECIFICITY OF THE BER ENZYMES

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The long-term goal of this project is to understand the structural basis for substrate specificity in two families of enzymes that play a critical role in the repair of ionizing radiation: the HhH-GPD Nth Superfamily and the Fpg Family. The fundamental hypothesis underlying the proposed work is that the molecular scaffold provided by any glycosylase superfamily or family member can support a wide variety of specificities for the excised base after substitution of a small number of residues. The specific aims are as follows: 1- We will solve the crystal structures of DNA-bound complexes of DNA glycosylases selected by phylogenetic/structural algorithms developed in Core A. We will study a subset of the Nth superfamily members where amino acid substitutions in the glycosylase result in altered substrate specificity. Similarly, we will examine complexes of a subset of naturally occurring DNA glycosylases that have similar sequences but divergent substrate specificities, such as EcNth and Micrococcus luteus UV endonuclease (M1Uve). 2- We will also solve the crystal structures of the DNA bound complexes of a subset of the Fpg Family members including the natural human orthologs of Fpg and Nei proteins, human NEI1 L, NEIL2, and NEIL3, in complex with their DNA substrate(s). Additionally, we will compare the liganded structm'e of NEIL1 with that ofArabidopsis thaliana Fpg, as the two enzymes share sequence similarity, but recognize very different substrates. Our structural studies will complement the available structural data and the substrate specificity studies in Project 1 and taken together they should provide a sound structural basis for a better understanding of substrate specificity in these classes of DNA glycosylases.