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 formation of high order structure of chromatin. HMGs with minor-groove binding 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 containing 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 assay and P-32-end labeled 27 bp AT-rich DNA, the dissociation constant of 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 length of AT-rich DNA. The possible DNA binding site of the HMG-I/DAT1 chimeric peptide is determined by footprinting experiments using a minor-groove cleaving agent ruthenium(III)-Schiff base complex and a 135-bp 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 chimeric peptide exhibits a preference for 5'-AAAT-3' of the AT-rich region. 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 design of short synthetic peptides for therapeutic applications.