The structures and relative stabilities of d(G · G) reverse hoogsteen, d(G·T) reverse wobble, and d(G · C) reverse watson-crick base-pairs in DNA crystals

Abstract

We have solved the structures of the homoduplex d(Gm5CGCGCG)2, and the heteroduplexes d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/d(CCGCGCG). The structures form six base-pairs of identical Z-DNA duplexes with single nucleotides overhanging at the 5′-ends. The overhanging nucleotide from one strand remains stacked and sandwiched between the blunt-ends of two adjacent Z-DNA duplexes, while the overhanging base of the opposing strand is extra-helical. The stacked and the extra-helical bases from adjacent duplexes pair to form a distorted d(G · G) reverse Hoogsteen base-pair in the d(Gm5CGCGCG)2 homoduplex, and d(G · T) reverse wobble and d(G · C) reverse Watson-Crick base-pairs in the d(GCGCGCG)/d(TCGCGCG) and d(GCGCGCG)/ d(CCGCGCG) heteroduplexes, respectively. Interestingly, only the d(G,T) and d(G · C) base-pairs were observed in the heteroduplexes, suggesting that both the d(G · T) reverse wobble and d(G · C) reverse Watson-Crick base-pairs are more stable in this crystal environment than the d(G · G) reverse Hoogsteen base-pair. To estimate the relative stability of the three types of reverse base-pairs, crystals were grown using various mixtures of sequences and their strand compositions analyzed by mass spectrometry. The d(G · C) reverse Watson-Crick base-pair was estimated to be more stable by ∼1.5 kcal/mol and the d(G · T) reverse wobble base-pair more stable by ∼0.5 kcal/mol than the d(G · G) reverse Hoogsteen base-pair. The step during crystallization responsible for discriminating between the strands in the crystal is highly cooperative, suggesting that it occurs during the initial nucleating event of crystal growth.