title: The substrate binding interface of alkylpurine DNA glycosylase AlkD.
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.
authors.affiliation: Department of Biological Sciences and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Elwood A
authors.initials: EA
authors.lastname: Mullins
authors.affiliation: Department of Biological Sciences and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Emily H
authors.initials: EH
authors.lastname: Rubinson
authors.affiliation: Department of Biological Sciences and Center for Structural Biology, Vanderbilt University, Nashville, TN 37232, USA. Electronic address: brandt.eichman@vanderbilt.edu.
authors.forename: Brandt F
authors.initials: BF
authors.lastname: Eichman
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D020134:Catalytic Domain; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C580784:N3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D045647:DNA Glycosylases; D000225:Adenine
keywords: 3-deaza-N3-methyladenine; 3d3mA; 3mA; 6-carboxyfluorescein; 7mG; ALK motif; Alkylpurine; Base excision repair; DNA glycosylase; FAM; HEAT repeat; N3-methyladenine; N7-methylguanine; Protein–DNA interaction; THF; tetrahydrofuran