American Chemical Society
bi952656o_si_001.pdf (189.7 kB)

Three-Dimensional Solution Structure of the N-Terminal Domain of DNA Polymerase β and Mapping of the ssDNA Interaction Interface,

Download (189.7 kB)
journal contribution
posted on 1996-05-21, 00:00 authored by Dingjiang Liu, Rajendra Prasad, Samuel H. Wilson, Eugene F. DeRose, Gregory P. Mullen
DNA polymerase β (β-Pol) consists of an N-terminal ssDNA binding domain with deoxyribose phosphodiesterase activity and a C-terminal domain with nucleotidyltransferase activity. The solution structure of the cloned N-terminal domain of β-Pol has been determined by multidimensional heteronuclear NMR using experimental restraints that included 1030 distances based on analysis of NOE connectivities, 68 φ, χ1, and χ2 torsion angles based on analysis of couplings, and 22 hydrogen bonds. Hydrogen bonds were assessed only within helices by the absence of saturation transfer from water at pH 6.7, by NOEs and JNHα couplings indicative of well-structured helices, and by 13Cα chemical shifts characteristic of helices. The root mean square deviation for heavy backbone atoms within the helices was 0.64 Å in 55 structures. The solution structure of the N-terminal domain is formed from four helices packed as two antiparallel pairs crossing at 50° in a V-like shape. The domain binds p(dT)8, a template analogue, as a 1:1 complex in 100 mM NaCl (KD = 10 μM). Analysis of the binding equilibria at increasing NaCl concentrations indicated that ionic contacts contribute to the complex. The binding interaction was mapped to one face of the domain by characterizing backbone 1H and 15N chemical shift changes. Assigned intermolecular NOEs from 2D NOESY support the assessment of the binding interface. The structure that forms the interaction surface includes an antiparallel helix-3−turn−helix-4 motif and residues adjacent to an Ω-type loop connecting helix-1 and helix-2. Sites appropriate for nucleotide contact on the structure are described. The mapped interaction interface for a ssDNA template is the first described for a DNA polymerase.