Faithful replication of DNA from one generation to the next is crucial for long-term species survival.
Genomic integrity in prokaryotes, archaea and eukaryotes is dependent on efficient and accurate catalysis by
multiple DNA polymerases.
Escherichia coli possesses five known DNA polymerases (Pol). DNA polymerase III holoenzyme is the major
replicative polymerase of the Escherichia coli chromosome (Kornberg, 1982). This enzyme contains two Pol III
cores that are held together by a t dimer (Studwell-Vaughan and O’Donnell, 1991). The core is composed of
three different proteins named α-, ε- and θ-subunit. The α-subunit, encoded by dnaE, contains the catalytic
site for DNA polymerisation (Maki and Kornberg, 1985), the ε-subunit, encoded by dnaQ, contains the 3′→5′
proofreading exonuclease (Scheuermann, et al., 1983) and the θ-subunit, encoded by hole, that has no
catalytic activity (Studwell-Vaughan, and O'Donnell, 1983).
The three-subunit α–ε–θ DNA pol III complex is the minimal active polymerase form purified from the DNA pol
III holoenzyme complex; these three polypeptides are tightly associated in the core (McHenry and Crow, 1979)
Despite a wealth of data concerning the properties of DNA polymerase III in vitro, little information is available
on the assembly in vivo of this complex enzyme.
In this study it is shown that the C-terminal region of the proofreading subunit is labile and that the ClpP
protease and the molecular chaperones GroL and DnaK control the overall concentration in vivo of ε.
Two α-helices (comprising the residues E311-M335 and G339-D353, respectively) of the N-terminal region of
the polymerase subunit were shown to be essential for the binding to ε.
These informations could be utilized to produce a conditional mutator strain in which proofreading activity
would be titrated by a a variant that can only bind e and that is polymerase-deficient. In this way the replication
of DNA made by DNA Pol-III holoenzyme would accordingly become error-prone.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:1593 |
| Date | 29 May 2009 |
| Creators | Bressanin, Daniela <1978> |
| Contributors | Hochkoeppler, Alejandro |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | Italian |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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