ABSTRACT
The transcriptional activity of many sequence-specific DNA binding proteins is directly regulated by posttranslational covalent modification. Although this form of regulation was first described nearly two decades ago, it remains poorly understood at a mechanistic level. The prototype for a transcription factor controlled by posttranslational modification is E. coli Ada protein, a chemosensor that both repairs methylation damage in DNA and coordinates the resistance response to genotoxic methylating agents. Ada repairs methyl phosphotriester lesions in DNA by transferring the aberrant methyl group to one of its own cysteine residues; this site-specific methylation enhances tremendously the DNA binding activity of the protein, thereby enabling it to activate a methylation-resistance regulon. Here, we report solution and X-ray structures of the Cys-methylated chemosensor domain of Ada bound to DNA. The structures reveal that both phosphotriester repair and methylation-dependent transcriptional activation function through a zinc- and methylation-dependent electrostatic switch.
Subject(s)
DNA Methylation , DNA Repair , DNA, Bacterial/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , O(6)-Methylguanine-DNA Methyltransferase/chemistry , Transcription Factors/chemistry , Transcriptional Activation , Crystallography, X-Ray , DNA Damage/drug effects , DNA Damage/physiology , DNA Methylation/drug effects , DNA Repair/physiology , DNA, Bacterial/metabolism , Escherichia coli Proteins/metabolism , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Protein Binding , Protein Structure, Tertiary , Regulon/physiology , Static Electricity , Transcription Factors/metabolism , Transcriptional Activation/physiologyABSTRACT
High-resolution structural studies of protein-DNA complexes have proven to be an invaluable means of understanding the diverse functions of proteins that manage the genome. Most of the structures determined to date represent proteins bound noncovalently to various DNA sequences or structures. Although noncovalent complexation is often adequate to study the structures of proteins that have robust, specific interactions with DNA, it is poorly suited to the study of transient intermediates in enzyme-catalyzed DNA processing reactions or of complexes that exist in multiple equilibrating forms. In recent years, strategies developed for the covalent trapping of protein-DNA complexes have begun to show promise as a window into an otherwise inaccessible world of structure.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Animals , Cross-Linking Reagents/pharmacology , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/metabolism , Disulfides/chemistry , Humans , Models, Molecular , N-Glycosyl Hydrolases/metabolism , Nucleic Acid Conformation , Protein Binding , Protein ConformationABSTRACT
Members of the HhH-GPD superfamily of DNA glycosylases are responsible for the recognition and removal of damaged nucleobases from DNA. The hallmark of these proteins is a motif comprising a helix-hairpin-helix followed by a Gly/Pro-rich loop and terminating in an invariant, catalytically essential aspartic acid residue. In this study, we have probed the role of this Asp in human 8-oxoguanine DNA glycosylase (hOgg1) by mutating it to Asn (D268N), Glu (D268E), and Gln (D268Q). We show that this aspartate plays a dual role, acting both as an N-terminal alpha-helix cap and as a critical residue for catalysis of both base excision and DNA strand cleavage by hOgg1. Mutation of this residue to asparagine, another helix-capping residue, preserves stability of the protein while drastically reducing enzymatic activity. A crystal structure of this mutant is the first to reveal the active site nucleophile Lys249 in the presence of lesion-containing DNA; this structure offers a tantalizing suggestion that base excision may occur by cleavage of the glycosidic bond and then attachment of Lys249. Mutation of the aspartic acid to glutamine and glutamic acid destabilizes the protein fold to a significant extent but, surprisingly, preserves catalytic activity. Crystal structures of these mutants complexed with an unreactive abasic site in DNA reveal these residues to adopt a sterically disfavored helix-capping conformation.
