Recognition properties of a sequence-specific DNA binding antibody

Biochemistry. 1998 Apr 28;37(17):6015-22. doi: 10.1021/bi9730792.


A sequence-specific DNA-binding antibody was previously generated by incorporating a 17 amino acid alpha-helix from the DNA-binding domain of the transcription factor TFEB into the HCDR3 site of a recombinant human Fab fragment. The recombinant DNA-binding antibody, called Fab-E box, binds the TFEB recognition sequence CACGTG (an E box site) with a 5-10-fold lower affinity than TFEB. Here, we have determined the precise kinetics of interaction of Fab-E box with DNA and show that the lower affinity of Fab-E box relative to TFEB for E box DNA is due to a higher dissociation rate. DNase I protection assays show Fab-E box physically interacts with one half-site of the E box. Additional DNA target sites of Fab-E box were identified by DNase I protection assays. A compilation of these binding sites indicates that the recognition elements for Fab-E box binding include a half-site of the E box, CAW, with an 8 bp consensus sequence identified as YNYYCAWW. Thus, the DNA determinants for Fab-E box recognition extend beyond one-half site of the E box sequence, with preferences for pyrimidines and A+T-rich sequences in the 5' and 3' outer regions of the binding site, respectively. Apparent dissociation constants of Fab-E box for a subset of these target DNA sequences are 5-10-fold greater than the DNA-binding affinity of the antibody with the E box site. Therefore, these results identify important DNA specificity determinants for high-affinity binding by Fab-E box.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibody Specificity / genetics
  • Base Sequence
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Binding Sites, Antibody* / genetics
  • Binding, Competitive / genetics
  • Binding, Competitive / immunology
  • DNA / immunology*
  • DNA / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism
  • Helix-Loop-Helix Motifs / genetics
  • Helix-Loop-Helix Motifs / immunology
  • Humans
  • Immunoglobulin Fab Fragments / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Neoplasm Proteins*
  • Transcription Factors*


  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • DNA-Binding Proteins
  • Immunoglobulin Fab Fragments
  • Neoplasm Proteins
  • TFEB protein, human
  • Transcription Factors
  • DNA