Steric and Electrostatic Effects in DNA Synthesis by the SOS-Induced DNA Polymerases II and IV of Escherichia coli

摘要

The SOS-induced DNA polymerases II and IV of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different parts in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogs of thymidine, in which substituted toluenes replace the polar thymine base. Comparison of thymine with nonpolar analogs was made to evaluate the importance of hydrogen bonding in the polymerase active sites, while comparisons among a set of variably-sized thymine analogs was used to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogs into a primer strand with high (wild-type) efficiency, while pol IV handled them with extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases’ sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.