Asymmetric 2 x 3 5'-GAA/AG-5' Internal Loop:
Stacked Adenosine Bracketed by Sheared G:A Base Pairs

Ko-Hsin Chin & Shan-Ho Chou*

Institute of Biochemistry

National Chung-Hsing University, Taichung, 40227, Taiwan

 

We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study the asymmetric internal loop of the highly conserved internal “5’-GAA:AG-5’ bubble” present in the linear single-stranded DNA genome of parvoviruses. This unusual motif contains a unpaired adenosine that is stacked between two bracketed sheared G:A pairs. Out of 30 initial embeds independently determined from the NMR data, 20 were found to converge to a single family of closely related structures with pair-wise r.m.s.d. values of 0.95 ± 0.15 Å. All sugar residues are in the normal C2’-endo conformation except for the single unpaired adenosine residue, which is in the unusual C3’-endo domain. Unlike the unusual BII-like GpA phosphodiesters in the adjacent G-A pairs, the GpA and ApA phosphodiesters in the GAA:GA motif exhibit the normal BI conformation, which is similar to those adopted in the (GGA)2 motif in the human centromere tandem repeat structure. The inserted adenosine acts as a wedge to kink the structure, causing a helix axis bending of about 20° at each insertion site. This well-structured “GAA:GA” motif that is present in the parvovirus genomes can explain the resistance of this region to the endonuclease susceptibility.