Stabilization of purine motif DNA triplex by a tetrapeptide from the binding domain of HMGBI protein

Abstract

DNA triple helix has attracted a lot of attention in the recent past because of their potential therapeutic applications to inhibit the transcription of genes associated with several human diseases. Therefore, studies on DNA triplex formation and stabilization have become very essential to understand the nature of DNA triplex both in quantitative and qualitative terms. We have studied the effect of a tetrapeptide, Pro-Lys-Arg-Trp (PKRW), containing the conserved sequence (PKR) of high mobility group (HMG) B1 DNA binding domain on the stability of purine motif DNA triplex. The triple stranded DNA complex (23R*R•Y) containing (GAA) repeats used in the present study is formed by: 5′-TCGCGAAGAAGAAGAAGAACGCT-3′, 5′-AGCGTTCTTCTTCTTCTTCGCGA-3′ and the third strand 5′-AAGAAGAAGAAGAAG-3′. The DNA triplex 23R*R•Y and its complex with PKRW were investigated by DNA-melting, UV-absorption, circular dichroism and fluorescence spectroscopy. The increased stability of 23R*R•Y triplex in presence of PKRW is supported by the following observations; (i) a marked increase in triplex melting temperature (ii) significant increase in the ellipticity of the characteristic negative band at 210 nm and (iii) a large change in free energy, Δ Δ G (Δ G of triplex-peptide complex – Δ G of free triplex), –9.6 kcal mol –1 on complexation with PKRW. Based on the above observations and fluorescence quenching data, we suggest a two stage model for the binding of PKRW to 23R*R•Y in which (a) PKRW binds to the minor groove of the 23R*R•Y with lysine and arginine making electrostatic interactions with phosphates of DNA, which is followed by (b) intercalation of tryptophan side chain between the A*A•T base triplets. We conclude that PKRW preferentially stabilizes Hoogsteen base pair to the Watson–Crick base pair in purine motif 23R*R•Y DNA triplex. It is tempting to speculate that HMGB1 plays an important role in recognizing the DNA triplex structures like H-DNA in the gene, which may have biological implications with respect to chromatin structure and its function in the cell.