UC Santa Cruz

Replicative DNA polymerases (DNAPs) are molecular motors responsible for high fidelity replication of the genome prior to each cellular division. These enzymes require two divalent cations (Me2+) to catalyze nucleotidyl transfer reactions. Many DNAPs have the ability to perform this chemical transformation in two active sites; (i) the polymerase active site where deoxyribonucleoside-triphosphate (dNTP) is selected and checked for template directed Watson-Crick base pairing prior to phosphodiester bond formation, and (ii) the exonuclease active site, where the 3ʹ primer terminal nucleotide can be excised in a manner consistent with proofreading the nascent DNA strand. Replicative DNAPs must choose the dNTP complementary to the template from a mixture of both dNTPs and ribonucleoside-triphosphates (rNTPs). A balance exists between substrate selection and proofreading, the primary mechanisms that ensure DNAP fidelity. Decades of biochemical research have identified several conserved residues associated with the activities of these two catalytic centers, using the highly processive DNAP from the bacteriophage phi29, structural studies implicated a residues Y254, Y390 & Y226, in the mechanism of translocation. Translocation occurs after nucleotide addition in the polymerase active site and is essential for processive DNAP replication. The translocation is a single nucleotide step that is required to move the DNAP along its DNA substrate, thus resetting the active site with the next template nucleotide poised for subsequent dNTP selection.

The kinetic complexity of replicative DNAPs requires an experimental method capable of distinguishing the multiple kinetic steps associated with DNAP activity. We developed the first such method for direct measurement of translocation, uniquely capable of making robust quantitative measurements with single nucleotide spatial precision and submillisecond temporal resolution. This method utilizes an α-Hemolysin nanopore embedded in a lipid bilayer which separates two wells of ionic solution to capture phi29 DNAP-DNA complexes in an electric field applied by a patch clamp amplifier. We have modeled the noncovalent kinetic transitions of translocation, nucleotide binding and primer strand transfer between the polymerase and exonuclease active sites. We demonstrated that in the pre-translocation state, which is analogous to the state the DNAP-DNA complex is in immediately following phosphodiester bond formation, a kinetic checkpoint exists where the polymerase can either send the primer terminus from the polymerase active site, in the pre-translocation state, to the exonuclease active site or it can retain the primer strand in the polymerase active site and translocate to the post-translocation state. If the polymerase transfers the primer strand to the exonuclease active site and the primer terminus is unedited then it can be returned from the exonuclease active site back to the polymerase active site in the pre-translocation state. If the polymerase translocates from the pre-translocation state to the post-translocation state then it is capable of binding to complimentary dNTP. Bound dNTP must dissociate before the polymerase can reverse translocate, from the post to the pre-translocation state.

We applied this experimental method to test several hypotheses regarding the mechanisms of high fidelity DNA replication. In the second chapter we describe the role of two conserved residues, Y226 & Y390, suggested by structural studies to be critical to the mechanism of translocation. Our data indicate that rather than being major determinants in the kinetic mechanism of translocation they are instead associated with active site assembly during dNTP substrate selection. In the third chapter we describe the kinetic mechanisms contributing to the stable incorporation of rNTPs into DNA by replicative DNAPs. In this study we systematically show that while the affinity for correctly base paired rNTP is considerably lower than it is for complementary dNTP, after incorporation of rNTP into the primer terminus the kinetic decision to edit or translocate is no more probable for complementary rNTP than dNTP. In chapter four we describe the role of divalent cations (Me2+) on the noncovalent transitions associated with DNAPs. Here we determined at submillimolar concentrations of Me2+ the kinetic pathway for primer strand transfer, from the pre-translocation state in the polymerase active site to and from the exonuclease active site, is composed of more than the two kinetic states identified in prior experiments with > 1mM Me2+. We also showed that across five orders of magnitude Me2+ causes a concentration dependent decrease in the rate of translocation from the pre to the post-translocation state and a concentration dependent increase in the rate of translocation from the post to the pre-translocation state. Also, we demonstrated that, in the presence of Ca2+, the presence of the primer terminal 3ʹOH does not contribute to the ground state stabilization of dNTP binding.