Title: The substrate binding interface of alkylpurine DNA glycosylase AlkD.

Authors: Mullins, Elwood A; Rubinson, Emily H; Eichman, Brandt F

Published In DNA Repair (Amst), (2014 Jan)

Abstract: Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture.

PubMed ID: 24286669

MeSH Terms: Adenine/analogs & derivatives*; Adenine/metabolism; Catalytic Domain; DNA Glycosylases/chemistry*; DNA Glycosylases/genetics; DNA Glycosylases/metabolism*; DNA Repair; DNA-Binding Proteins/chemistry*; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism*; Guanine/analogs & derivatives*; Guanine/metabolism; Models, Molecular; Mutation; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Substrate Specificity