Networked communication between polymerase and exonuclease active sites in human mitochondrial DNA polymerase

High fidelity human mitochondrial DNA polymerase (Pol γ) contains two active sites, a DNA polymerization site (pol) and a 3′-5′ exonuclease site (exo) for proofreading. Although separated by 35 A, coordination between the pol and exo sites is crucial to high fidelity replication. The biophysical mechanisms for this coordination are not completely understood. To understand the communication between the two active sites, we used a statistical-mechanical model of the protein ensemble to calculate the energetic landscape and local stability. We compared a series of structures of Pol γ, complexed with primer/template DNA, and either a nucleotide substrate or a series of nucleotide analogues, which are differentially incorporated and excised by pol and exo activity. Despite the nucleotide or its analogues being bound in the pol, Pol γ residue stability varied across the protein, particularly in the exo domain. This suggests that substrate presence in the pol can be “sensed” in the exo domain. Consistent with th...