Trapping distinct structural states of a protein/DNA interaction through disulfide crosslinking.
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 approximately 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.
He
Chuan
C
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Verdine
Gregory L
GL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides; D029968:Escherichia coli Proteins; D008745:Methylation; D015336:Molecular Probe Techniques; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011485:Protein Binding; D014157:Transcription Factors
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D014157:Transcription Factors; D004247:DNA; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase