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.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Ozawa K,Jergic S,Park AY,Dixon NE,Otting Gdoi
10.1093/nar/gkn489subject
Has Abstractpub_date
2008-09-01 00:00:00pages
5074-82issue
15eissn
0305-1048issn
1362-4962pii
gkn489journal_volume
36pub_type
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