Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion

Abstract

Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

“…However,r ecent timeresolved X-ray crystallographic studies of DNA polymerases, including the Xf amily polymerasesP ol b andP ol m,s uggest the involvement of at hird metal ion. [20] Although this has not been observed in the case of the TdT as of yet, the participation of at hird metal ion could help to explain the movements of metal A. [51]…”

“…These enzymes belong to one of six families (A, B, C, D, X, and Y) that have been established according to their sequence homology and phylogenetic relationships . Members of these families are devoted to particular roles and biological functions that are reflected by specific cellular localizations, a number of DNA substrates and nucleotide specificities, varying processivities, and often different kinetic mechanisms . Polymerases constituting the A family are involved in DNA repair and replication and prominent members include the Klenow fragment of Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, and the T7 RNA and DNA polymerases.…”

“…The TdT is, in many aspects, a unique polymerase: the TdT catalyzes the addition of nucleotides in a template‐independent manner, prefers ssDNA to dsDNA, and is unapt to copy templates …”

“…Overall, the TdT appears to adopt a typical two‐metal‐ion mechanism, in which the metal ions are coordinated to three conserved aspartic acid residues that mainly position and activate the free 3′‐OH group of the primer for in‐line nucleophilic attack on the P α atom of the incoming nucleotide. However, recent timeresolved X‐ray crystallographic studies of DNA polymerases, including the X family polymerases Pol β and Pol μ, suggest the involvement of a third metal ion . Although this has not been observed in the case of the TdT as of yet, the participation of a third metal ion could help to explain the movements of metal A …”

“…[17c, 19] Members of these familiesa re devoted to particularr oles and biological functions that are reflected by specific cellular localizations, an umber of DNA substrates and nucleotide specificities, varying processivities, and often different kinetic mechanisms. [20] Polymerases constituting the A family are involved in DNA repair and replication and promi-The terminal deoxynucleotidyl transferase (TdT) belongs to the Xf amily of DNA polymerases. This unusual polymerase catalyzes the template-independent addition of randomn ucleotides on 3'-overhangs during V(D)J recombination.…”