Abstract
The N-terminal domain of the Escherichia coli Ada protein (N-Ada) repairs methyl phosphotriesters in DNA through a zinc-mediated transfer to Cys38 of the protein. Methylation of Cys38 enhances the sequence-specific DNA affinity of N-Ada by ∼1000-fold, thereby enabling the protein to activate the genes of a methylation-resistance regulon. It is of interest to understand the structural basis for metalloactivated methyl transfer and methylation-dependent enhancement of DNA binding activity. Although recent progress has been made on the structural front, efforts to develop a complete picture of N-Ada structure/function have been hampered by the inability to prepare homogeneous protein/DNA complexes representing different states of the unmethylated protein. Here, we describe the development of an approach to trap both sequence-specific and nonsequence-specific DNA recognition complexes of N-Ada through formation of an intermolecular disulfide crosslink between the protein and DNA.
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