NMR solution structure of the θ subunit of DNA polymerase III from Escherichia coli

Abstract

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits~a, E, and u!. The u subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the u subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, u was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3 J HNa coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments~Gln10-Asp19, Glu38-Glu43, and His47-Glu54!, one short extended segment Pro34-Ala37!, and a long loop~Ala20-Glu29!, of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of u is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the E subunit have identified a loop region~Ala20-Glu29! in u as the site of association with E.

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the ε and θ subunits of DNA polymerase III

“…That the mass spectrum was unchanged after treatment of 186-with 9 M NH 4 OAc for 1 h indicates that the complex is stable under these conditions. This stability at such high salt concentrations suggests that electrostatic interactions do not play a major role in the overall stability of 186-, and is consistent with NMR spectroscopic studies that indicate that contacts between the two subunits largely involve hydrophobic residues (Keniry et al 2000;DeRose et al 2003).…”

“…The and 186 subunits of DNA polymerase III were overproduced in E. coli and purified as described previously (Keniry et al 2000;Hamdan et al 2002a). These protein samples and the isolated 186-complex had previously been characterized by mass spectrometry, giving masses in good agreement with the calculated values of 8848.0 (), 20,586.9 (186), and 29,434.9 (186-) (Hamdan et al 2002a).…”

“…Although there is no high-resolution structure yet available for the 186-complex, we have reported the crystal structure of 186 (Hamdan et al 2002b) and the solution structure of (Keniry et al 2000). NMR chemical shift mapping experiments in the latter study suggested that a series of small hydrophobic residues on the external face of the first helix of (residues 21-27, AAAGVAF) are involved in its association with 186, and this has been confirmed in the more recent NMR structure of in the 186-complex (M. Keniry, pers.…”

“…The instability of 186 (toward aggregation) was revealed by the dramatic diminution of ion current over narrow concentration ranges of organic solvents. On the other hand, although (or perhaps because) is known to be a poorly structured protein (Keniry et al 2000), it proved to be remarkably resilient under these conditions. It was also shown to protect 186 from the effects of these solvents, and the 186-complex was shown to form rapidly and to dissociate very slowly in solutions containing high concentrations of alkanols at room temperature.…”

Replisome Dynamics during Chromosome Duplication

“…Deletion of the gene encoding θ (holE ) does not affect cell viability (142), but in vitro and in vivo experiments imply a slight stabilization and stimulation of the ε exonuclease activity by θ (151, 155). The solution structure of θ reveals a chain fold that resembles the DNA-interacting domain of eukaryotic DNA polymerase β (78). However, θ has not been demonstrated to bind DNA and does not appear to directly interact with the α subunit (151).…”