The structure and kinetics of an unusual DNA polymerase I from the thermophilic bacterium, Rhodothermus marinus

DNA polymerase I employs a multistep mechanism for sorting correctly paired nucleotides from mismatches. We aim to characterize reaction intermediates during nucleotide selection to better understand how this class of enzymes achieves high DNA replication fidelity. DNA polymerase I from R. marinus contains an unusual and disruptive proline in the mobile O‐ helix near the active site. To characterize this unusual enzyme, we solved the structure of the large (5′‐to‐3′ deficient) fragment of the R. marinus DNA polymerase I (RF) to 2.95 Å ( R = 0.246) using multi‐wavelength anomalous dispersion. Alignment with homologous Escherichia coli Klenow Fragment (KF) DNA polymerase confirmed that the active sites of each structural domain were conserved. In order to study the polymerase activity in isolation, we eliminated the 3′‐to‐5′ exonuclease activity using site‐directed mutagenesis of an aspartic acid involved in binding DNA (D497A). Unexpectedly, mutation of an aspartic acid involved in binding a catalytic magnesium ion (D421A in RF) failed to abolish exonuclease activity despite its ability to do so in the KF (D424A). Enzyme kinetic assays of the 3′‐to‐5′ exonuclease and polymerase activities of RF and its mutants indicate that RF is a highly unusual member of the DNA polymerase I family. Work funded by University of Richmond School of Arts & Sciences.