DESIGN AND CHARACTERIZATION OF A SHORT HMG-I DAT1 PEPTIDE THAT BINDS SPECIFICALLY TO THE MINOR-GROOVE OF DNA/

High mobility group protein (HMG) is known to be involved in the forma tion of high order structure of chromatin. HMGs with minor-groove bind ing ability in the AT-rich DNA region play a vital role in controlling gene transcription activity. In this report, a 18-residue HMG-I/DAT1 chimeric peptide, PRGRPKGKTLREPRGRPY, was designed and synthesized con taining two repetitive PRGRP units and a linking peptide, KGKTLRE, as a targeted DNA-binding peptide. The segment PRGRP is derived from HMG- I while KGKTLRE is from the DAT1 peptide. Using gel-mobility shift ass ay and P-32-end labeled 27 bp AT-rich DNA, the dissociation constant o f this chimeric peptide was found to be 4.7 x 10(-6) M, that is, 10(4) times stronger than that of the PRGRP segment stand alone (> 10(-2) M ). In addition, the binding constant was found to increase with the le ngth of AT-rich DNA. The possible DNA binding site of the HMG-I/DAT1 c himeric peptide is determined by footprinting experiments using a mino r-groove cleaving agent ruthenium(III)-Schiff base complex and a 135-b p P-32-5'-end-labeled DNA restriction fragment of Hind III/Rsa I from plasmid pBR322 DNA. The major pattern protected by the HMG-I/DAT1 chim eric peptide exhibits a preference for 5'-AAAT-3' of the AT-rich regio n. Therefore, this novel design HMG-I/DAT1 chimeric peptide possesses not only a high affinity to AT-rich DNA but also the sequence-specific binding in the minor groove of DNA, which may further lead to the des ign of short synthetic peptides for therapeutic applications.