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Determining the kinetics of DnaE, an enzyme for DNA replication in S. aureus (583.8)

DnaE is an DNA polymerase of the C family enzymes. It is used in DNA replication in S.aureus bacterium where this enzyme has been found to be crucial for lagging strand replication. We conducted pre‐steady state and steady state assays to look at the kinetic nature of DnaE in the replicative pathway. The pathway begins with DnaE binding to DNA substrate. Pre‐steady state condition assays showed that the affinity of the enzyme to bind to DNA was kDDNA=7±2nM. Single‐turn over condition assays gave a value of kDdNTP=30±2μM for the affinity of a nucleotide to be added to the duplex with enzyme complex. After this step the replicative polymerase may bind to other DNA to continue replication. A koff=0.1145sec‐1 rate was found for the disassociation of the enzyme from the binary structure. Replication with DnaE continues at a steady state rate of kcat=0.1 sec‐1.These values helped to determine that the rate at which the enzyme dissociates from the DNA duplex must be the rate limiting step in DnaE replication. Comparing these values to PolC, the enzyme that replicates the leading strand in S.aureus, and other C family polymerases may help answer why this enzyme must begin lagging strand replication at the primer.