The proofreading exonuclease subunit epsilon of Escherichia coli DNA polymerase III is tethered to the polymerase subunit alpha via a flexible linker.
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.
Ozawa
Kiyoshi
K
Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia.
Jergic
Slobodan
S
Park
Ah Young
AY
Dixon
Nicholas E
NE
Otting
Gottfried
G
D000595:Amino Acid Sequence; D002474:Cell-Free System; D004258:DNA Polymerase III / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; C505238:holE protein, E coli; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C468316:dnaQ protein, E coli; D005090:Exodeoxyribonucleases