Overexpression and purification of trimeric τ-α-ε-θ sub-assembly of E. coli DNA Polymerase III

The coordinated replication of leading and lagging DNA strands can be fulfilled by bi-functional DNA Polymerases (DNA Pols), featuring two independent catalytic sites. In E. coli, the DNA Pol competent in genome replication, i.e. DNA Pol III, is generally thought to be an asymmetric dimer, comprising 10 different subunits (McHenry, 2011). Among these, α,ε, and θ associate to form the catalytic core, with α subunit providing the 5’-3’ Pol activity, and ε responsible for 3’-5’ exonuclease (proofreading) activity. Two independent catalytic cores can associate via the DnaX complex, composed of the τ, γ, ψ, χ, δ, and δ’ subunits, assembled according to τ2γ1 ψ1χ1δ1δ’1 stoichiometry. In addition, high processivity is conferred to E. coli Pol III by the β subunit, acting as a clamp on DNA and interacting with α. Interestingly, the γ subunit is coded by the same gene (dnaX) yielding τ. A frameshift in translation of dnaX message is indeed responsible for early termination, producing a C-ter truncated form of τ, i.e. γ subunit (Flower and McHenry, 1990). Moreover, it was demonstrated that using a mutant form of dnaX, devoid of the frameshift motif, only the τ subunit is expressed (Blinkowa and Walker, 1990). Due to the deficiency of the τ C-ter region, γ is not competent in binding α. This is relevant when considering that τ self-interacts and contacts α, therefore representing the subunit bridging two catalytic cores, whose parallel action withstands leading and lagging strand replication. Recently, a trimeric replicase containing 3 τ subunits and 3 catalytic cores was assembled in vitro, and it was shown that the 3 active sites of this replicase are able to exert their Pol function simultaneously (McInerney et al., 2007). To assess whether a trimeric replicase can be assembled in vivo, and to further test the τ/core stoichiometry, we co-expressed in E. coli α, ε, and θ subunits, along with a dnaX variant unable to prdouce γ. With this aim, we cloned into the pBAD plasmid the dnaE gene (coding for α), and into the pGOOD vector (Conte et al., 2011) the dnaX-γless, dnaQ, and holE genes (coding for τ, ε, and θ, subunits, respectively). E. coli was co-transformed with these two plasmids and the overexpression of α, τ, ε, and θ was triggered by the addition of arabinose and IPTG to the growth medium. Soluble proteins were then extracted from overexpressing cells, and the purification of the ταεθ complex was achieved by means of standard chromatographic techniques. To our surprise, no dimeric replicase (τ2α2ε2θ2) was detected in protein extracts. On the contrary, trimeric Polymerase (τ3α3ε3θ3) was readily purified, along with an excess of catalytic core. Polymerase activity was then assayed using the trimeric replicase, and the observed activity was compared to that of purified α subunit. In this way, the 3 catalytic cores of τ3α3ε3θ3 were found to exert their function simultaneously. At the present time we are investigating whether two catalytic cores of the trimeric replicase cooperate in lagging strand replication.