Recognition properties of a sequence-specific DNA binding antibody

Biochemistry
J F LeBlancP Ghazal

Abstract

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...Continue Reading

References

Feb 1, 1985·The Journal of Clinical Investigation·R S Schwartz, B D Stollar

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Citations

Dec 28, 1999·Journal of Molecular Recognition : JMR·D G Myszka
Sep 13, 2001·Bioorganic & Medicinal Chemistry·J A Beckingham, G D Glick
Nov 24, 2004·Protein Science : a Publication of the Protein Society·Hulin JinOscar R Burrone
Mar 1, 2006·Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics·Jorge M O FernandesIan A Johnston
Mar 2, 2012·The Journal of Biological Chemistry·Olga A TimofeevaAnatoly Dritschilo
Mar 30, 2001·The Journal of Biological Chemistry·M L CeruttiG de Prat-Gay
Mar 9, 2021·Frontiers in Physiology·Manman LiLiming Yang

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