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Recognition properties of a sequence-specific DNA binding antibody

LeBlanc, James F., McLane, Kathryn E., Parren, Paul W. H. I., Burton, Dennis R. and Ghazal, Peter ORCID: 1998. Recognition properties of a sequence-specific DNA binding antibody. Biochemistry 37 (17) , pp. 6015-6022. 10.1021/bi9730792

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A sequence-specific DNA-binding antibody was previously generated by incorporating a 17 amino acid α-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.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Medicine
Publisher: American Chemical Society
ISSN: 0006-2960
Last Modified: 23 Oct 2022 14:27

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