Elucidation of the ε−θ Subunit Interface of Escherichia coli DNA Polymerase III by NMR Spectroscopy

• Published in: Biochemistry (Easton) Vol. 42; no. 13; pp. 3635 - 3644
• Main Authors: DeRose, Eugene F, Darden, Thomas, Harvey, Scott, Gabel, Scott, Perrino, Fred W, Schaaper, Roel M, London, Robert E
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08-04-2003
• Subjects: Amino Acid Sequence; Binding Sites; Catalytic Domain; DNA Replication; Enzyme Stability; Escherichia coli - enzymology; Exodeoxyribonuclease V; Exodeoxyribonucleases - chemistry; Exodeoxyribonucleases - genetics; Exonucleases - chemistry; Models, Molecular; Molecular Sequence Data; Mutation - genetics; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Conformation; Protein Subunits; Sequence Homology, Amino Acid
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0205451

The DNA polymerase III holoenzyme (HE) is the primary replicative polymerase of Escherichia coli. The epsilon (ε) subunit of HE provides the 3‘→5‘ exonucleolytic proofreading activity for this complex. ε consists of two domains:  an N-terminal domain containing the proofreading exonuclease activity (residues 1−186) and a C-terminal domain required for binding to the polymerase (α) subunit (residues 187−243). In addition to α, ε also binds the small (8 kDa) theta (θ) subunit. The function of θ is unknown, although it has been hypothesized to enhance the 3‘→5‘ exonucleolytic proofreading activity of ε. Using NMR analysis and molecular modeling, we have previously reported a structural model of ε186, the N-terminal catalytic domain of ε [DeRose et al. (2002) Biochemistry 41, 94]. Here, we have performed 3D triple resonance NMR experiments to assign the backbone and Cβ resonances of [U-2H,13C,15N] methyl protonated ε186 in complex with unlabeled θ. A structural comparison of the ε186−θ complex with free ε186 revealed no major changes in secondary structure, implying that the overall structure is not significantly perturbed in the complex. Amide chemical shift comparisons between bound and unbound ε186 revealed a potential binding surface on ε for interaction with θ involving structural elements near the ε catalytic site. The most significant shifts observed for the ε186 amide resonances are localized to helix α1 and β-strands 2 and 3 and to the region near the beginning of α-helix 7. Additionally, a small stretch of residues (K158−L161), which previously had not been assigned in uncomplexed ε186, is predicted to adopt β-strand secondary structure in the ε186−θ complex and may be significant for interaction with θ. The amide shift pattern was confirmed by the shifts of aliphatic methyl protons, for which the larger shifts generally were concentrated in the same regions of the protein. These chemical shift mapping results also suggest an explanation for how the unstable dnaQ49 mutator phenotype of ε may be stabilized by binding θ.