Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.
In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000077209:Decitabine; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D013698:Templates, Genetic; D014961:X-Ray Diffraction
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D000077209:Decitabine; D003596:Cytosine; D004247:DNA; D001374:Azacitidine