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PMID:18663010

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Citation

Ozawa, K, Jergic, S, Park, AY, Dixon, NE and Otting, G (2008) The proofreading exonuclease subunit epsilon of Escherichia coli DNA polymerase III is tethered to the polymerase subunit alpha via a flexible linker. Nucleic Acids Res. 36:5074-82

Abstract

Escherichia coli DNA polymerase III holoenzyme is composed of 10 different subunits linked by noncovalent interactions. The polymerase activity resides in the alpha-subunit. The epsilon-subunit, which contains the proofreading exonuclease site within its N-terminal 185 residues, binds to alpha via a segment of 57 additional C-terminal residues, and also to theta, whose function is less well defined. The present study shows that theta greatly enhances the solubility of epsilon during cell-free synthesis. In addition, synthesis of epsilon in the presence of theta and alpha resulted in a soluble ternary complex that could readily be purified and analyzed by NMR spectroscopy. Cell-free synthesis of epsilon from PCR-amplified DNA coupled with site-directed mutagenesis and selective 15N-labeling provided site-specific assignments of NMR resonances of epsilon that were confirmed by lanthanide-induced pseudocontact shifts. The data show that the proofreading domain of epsilon is connected to alpha via a flexible linker peptide comprising over 20 residues. This distinguishes the alpha : epsilon complex from other proofreading polymerases, which have a more rigid multidomain structure.

Links

PubMed PMC2528190 Online version:10.1093/nar/gkn489

Keywords

Amino Acid Sequence; Cell-Free System; DNA Polymerase III/chemistry; DNA-Directed DNA Polymerase/chemistry; Escherichia coli Proteins/chemistry; Exodeoxyribonucleases/chemistry; Molecular Sequence Data; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Tertiary; Sequence Homology, Amino Acid

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:DPO3E

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P10443

F

Seeded From UniProt

complete

ECOLI:DPO3E

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P0ABS8

F

Seeded From UniProt

complete

ECOLI:HOLE

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P03007

F

Seeded From UniProt

complete

ECOLI:DPO3A

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P03007

F

Seeded From UniProt

complete

See also

References

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