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A New Role for the Proofreader in Bacterial DNA Replication
Author(s) -
Dixon Nicholas E.,
Jergic Slobodan
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.739.4
Subject(s) - processivity , dna polymerase , dna clamp , dna replication , polymerase , primer (cosmetics) , dna polymerase delta , biology , dna polymerase ii , genetics , chemistry , microbiology and biotechnology , biophysics , dna , reverse transcriptase , polymerase chain reaction , gene , organic chemistry
The DNA polymerase III holoenzyme (Pol III HE), the E. coli chromosomal replicase, is made up of 2 (or 3) αεθ core polymerases, 3 dimeric β sliding clamps, and the 7‐subunit clamp loader that loads the clamp onto primer‐templates. When bound to the clamp, the Pol III core becomes fast (~750 nt/s) and processive (>50 kb) due to the α‐β interaction. The ε subunit is the proofreading 3′→5′ exonuclease that interacts with α and greatly increases the fidelity of Pol III HE. Genetic studies 20 years ago suggested that ε has an essential role in replication beyond its contribution to fidelity, and we have previously also shown that it is attached to α via a flexible linker that permits it to sample a relatively large space around α in the replicase. Here, we present new functional assays (Pol III strand displacement and primer extension under difficult conditions) that expose the additional role of ε in DNA replication. Bioinformatics indicated it may be mediated by a new interaction with the β clamp. We further utilized SPR and ESI‐MS to obtain direct evidence for a physical interaction between ε and β both in isolation and within the Pol III‐clamp complex. Single molecule DNA replication assays revealed the role of the relatively weak yet important ε‐β contact in stabilization of the polymerase on the DNA template.