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Title
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Three-dimensional solution structure of the N-terminal domain of DNA polymerase beta and mapping of the ssDNA interaction interface.
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Authors
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D.Liu,
R.Prasad,
S.H.Wilson,
E.F.DeRose,
G.P.Mullen.
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Ref.
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Biochemistry, 1996,
35,
6188-6200.
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PubMed id
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Abstract
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DNA polymerase beta (beta-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 beta-Pol has been determined by multidimensional heteronuclear NMR
using experimental restraints that included 1030 distances based on analysis of
NOE connectivities, 68 phi, chi 1, and chi 2 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
JNH alpha couplings indicative of well-structured helices, and by 13C alpha
chemical shifts characteristic of helices. The root mean square deviation for
heavy backbone atoms within the helices was 0.64 A in 55 structures. The
solution structure of the N-terminal domain is formed from four helices packed
as two antiparallel pairs crossing at 50 degrees 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
microM). 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 omega-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.
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