Structural and Biochemical Studies of Alkylpurine DNA Glycosylase AlkD

Abstract

Alkylating agents covalently modify DNA bases to generate a chemically diverse set of lesions including cytotoxic N3-methyladenine (3mA) bases and mutagenic 1,N6-ethenoadenine (εA) and N7-methylguanine (7mG). To maintain genomic integrity, these lesions are repaired by the base excision repair pathway which is initiated by DNA glycosylases, enzymes that locate and excise modified nucleobases by cleaving the N-glycosylic bond. The basis for glycosylase specificity toward N3- and N7-alkylpurines remains unclear. This dissertation is a structure-function analysis of a recently discovered 3mA DNA glycosylase, Bacillus cereus AlkD. The crystal structure of AlkD reveals that AlkD represents a sixth structural superclass of DNA glycosylases and is the first structural example of a HEAT-like repeat protein that has a defined catalytic activity and binds DNA. Crystal structures of the enzyme alone and in complex with DNAs containing alkylated, mismatched, and abasic nucleotides reveal that unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation rather than an active site pocket on the protein, and provides the first illustration for how hydrolysis of unstable N3- and N7-alkylated bases can be facilitated by increased lifetime out of the DNA helix. The AlkD-DNA structures and supporting biochemical analysis of base flipping and catalysis also reveal how AlkD’s tandem HEAT-repeats distort the DNA backbone to sense energetic differences in non-Watson-Crick base pairs without duplex intercalation, and support the idea that glycosylase specificity toward N3- and N7-alkylpurines may result from intrinsic instability of the modified base and not from direct enzyme functional group chemistry. Using the crystal structures as a guide, a comprehensive mutational analysis of DNA binding and catalytic activity has been performed to examine the role of selected residues. In addition, AlkD has been utilized in a pilot study for the development of a mass spectrometry method to simultaneously quantify the wide spectrum of alkylated bases liberated from DNA by alkylpurine DNA glycosylases. Thus, the AlkD protein is a useful model system to investigate enzymatic removal of N3- and N7-methylpurines from DNA, and provides the first glimpse into nucleic acid binding by a HEAT repeat architecture.