scholarly journals Structure of the θ Subunit of Escherichia coli DNA Polymerase III in Complex with the ε Subunit

2006 ◽  
Vol 188 (12) ◽  
pp. 4464-4473 ◽  
Author(s):  
Max A. Keniry ◽  
Ah Young Park ◽  
Elisabeth A. Owen ◽  
Samir M. Hamdan ◽  
Guido Pintacuda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Active Site ◽  
Dimensional Structure ◽  
Resonance Spectroscopy ◽  
Dna Polymerase Iii ◽  
Pseudocontact Shifts ◽  
Catalytic Core ◽  
Lanthanide Ion ◽  
Polymerase Iii

ABSTRACT The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε186 on θ was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of θ are located about 15 Å or farther from the lanthanide ion in the active site of ε186, in agreement with the extensive biochemical data for the θ-ε system.

scholarly journals Nuclear Magnetic Resonance Solution Structure of the Escherichia coli DNA Polymerase III θ Subunit

2005 ◽  
Vol 187 (20) ◽  
pp. 7081-7089 ◽  
Author(s):  
Geoffrey A. Mueller ◽  
Thomas W. Kirby ◽  
Eugene F. DeRose ◽  
Dawei Li ◽  
Roel M. Schaaper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nuclear Magnetic Resonance ◽  
Magnetic Resonance ◽  
Dna Polymerase ◽  
Solution Structure ◽  
Conformational Exchange ◽  
Dna Polymerase Iii ◽  
Catalytic Core ◽  
Polymerase Iii ◽  
Nuclear Magnetic

ABSTRACT The catalytic core of Escherichia coli DNA polymerase III holoenzyme contains three subunits: α, ε, and θ. The α subunit contains the polymerase, and the ε subunit contains the exonucleolytic proofreading function. The small (8-kDa) θ subunit binds only to ε. Its function is not well understood, although it was shown to exert a small stabilizing effect on the ε proofreading function. In order to help elucidate its function, we undertook a determination of its solution structure. In aqueous solution, θ yielded poor-quality nuclear magnetic resonance spectra, presumably due to conformational exchange and/or protein aggregation. Based on our recently determined structure of the θ homolog from bacteriophage P1, named HOT, we constructed a homology model of θ. This model suggested that the unfavorable behavior of θ might arise from exposed hydrophobic residues, particularly toward the end of α-helix 3. In gel filtration studies, θ elutes later than expected, indicating that aggregation is potentially responsible for these problems. To address this issue, we recorded 1H-15N heteronuclear single quantum correlation (HSQC) spectra in water-alcohol mixed solvents and observed substantially improved dispersion and uniformity of peak intensities, facilitating a structural determination under these conditions. The structure of θ in 60/40 (vol/vol) water-methanol is similar to that of HOT but differs significantly from a previously reported θ structure. The new θ structure is expected to provide additional insight into its physiological role and its effect on the ε proofreading subunit.

scholarly journals Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

2012 ◽  
Vol 523 (2) ◽  
pp. 135-143 ◽  
Author(s):  
Emanuele Conte ◽  
Gabriele Vincelli ◽  
Roel M. Schaaper ◽  
Daniela Bressanin ◽  
Alessandra Stefan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Catalytic Core ◽  
Polymerase Iii ◽  
Pol Iii

Proteolysis of the proofreading subunit controls the assembly of Escherichia coli DNA polymerase III catalytic core

2009 ◽  
Vol 1794 (11) ◽  
pp. 1606-1615 ◽  
Author(s):  
Daniela Bressanin ◽  
Alessandra Stefan ◽  
Fabrizio Dal Piaz ◽  
Stefano Cianchetta ◽  
Luca Reggiani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Catalytic Core ◽  
Polymerase Iii

scholarly journals The interaction of DNA polymerase III and the product of the Escherichia coli mutator gene, mutD.

1984 ◽  
Vol 259 (9) ◽  
pp. 5567-5573
Author(s):  
R DiFrancesco ◽  
S K Bhatnagar ◽  
A Brown ◽  
M J Bessman
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Mutator Gene ◽  
Polymerase Iii

scholarly journals Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1039-1044 ◽  
Author(s):  
I J Fijalkowska ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Alpha Subunit ◽  
Sequence Motifs ◽  
Dna Polymerase Iii ◽  
Conserved Sequence ◽  
Polymerase Iii ◽  
Dna Replication Errors

Abstract The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

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