Conformational microheterogeniety in a DNA double helix: Structure of restriction endonuclease Bam H1 recognition site

Abstract

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC) 2 in solution at 19° is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2′/H2″, H3′ reveal that the nucleotide units G2, A3 and C6 adopt (C3′- endo , χ = 200°–220°) conformation while G1, T4 and C5 exhibit (C2′- endo , χ = 240°–260°) conformation. NMR data clearly suggest that the two strands of d(GGATCC) 2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC) 2. The important features are: (i) G1–G2 stack, the site of cleaveage, shows an alternation in sugar pucker i.e. C2′- endo , C3′- endo as in a B-A junction, (ii) G2–A3 stack adopts a mini A-DNA, both the sugars being C3′- endo , (iii) A3–T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3′- endo , C2′- endo , (iv) T4–C5 stack adopts a mini B-DNA both the sugars being C2′- endo and (v) C5–C6 stack exhibits a B-A junction with C2′- endo , C3′- endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeniety with a structural two fold, is present in the Bam H1 recognition site.