RECOGNITION PROPERTIES OF A SEQUENCE-SPECIFIC DNA-BINDING ANTIBODY

A sequence-specific DNA-binding antibody was previously generated by i ncorporating a 17 amino acid alpha-helix from the DNA-binding domain o f the transcription factor TFEB into the HCDR3 site of a recombinant h uman 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 prec ise kinetics of interaction of Fab-E box with DNA and show that the lo wer affinity of Fab-E box relative to TFEB for E box DNA is due to a h igher dissociation rate. DNase I protection assays show Fab-E box phys ically interacts with one half-site of the E box. Additional DNA targe t sites of Fab-E box were identified by DNase I protection assays. A c ompilation of these binding sites indicates that the recognition eleme nts 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 deter minants for Fab-E. box recognition extend beyond one-half site of the E box sequence, with preferences for pyrimidines and A+T-rich sequence s in the 5' and 3' outer regions of the binding site, respectively. Ap parent dissociation constants of Fab-E box for a subset of these targe t DNA sequences are 5-10-fold greater than the DNA-binding affinity of the antibody with the E box site. Therefore, these results identify i mportant DNA specificity determinants for high-affinity binding by Fab -E box.