Open Access Dissertation
Date of Award
Louis J. Romano
DNA synthesis is critical to cell survival, and the enzyme DNA polymerase has had a great deal of time to evolve efficient strategies to perform synthesis. A variety of effects are induced by the identity of the incoming dNTP, and its ability to effectively base pair with the templating base. The presence of the next correct dNTP is known to induce a conformational change of the polymerase and lead to an increase in KF-DNA binding. Conversely, the presence of an incorrect dNTP is shown to lead to a destabilization and reduction in the levels of KF-DNA complexes formed. Using surface plasmon resonance and an assay that directly examines the impact of the identity of the incoming dNTP on formation of KF-DNA complexes, we present evidence for the formation of a destabilized complex in the presence of incorrectly pairing dNTPs. We present and discuss the implications of a new model for DNA synthesis that involves a dNTP selection cycle. This selection cycle shows how polymerases aid in the removal of mispairing dNTPs from their active sites in an effort to actively seek a correctly pairing template-dNTP for catalysis. Polymerases have evolved to efficiently utilize this method of dNTP selection to speed up dNTP selection.
Bulky DNA adducts such as benzo[a]pyrene interfere with replication by forming structures within the polymerase that preclude the productive binding of dNTPs, and/or inhibit this conformational change. Surface plasmon resonance was used to study the effect various dNTPs have upon Klenow fragment binding to several (-)-trans-anti-B[a]P-N2-dG adducted primer-templates. Binding constants were determined for Klenow fragment binding to primer templates containing either a correctly paired C:G-B[a]P, mismatched T:G-B[a]P, or mismatched G:G-B[a]P positioned at the -1 position, in the presence and absence of various dNTPs and rNTPs. The presence of the (-)-trans-anti-B[a]P-N2-dG adduct interferes with the formation of a stable closed ternary complex. Moreover, the addition of any dNTP favoured formation of a destabilized ternary complex that rapidly dissociates to free polymerase and DNA. In addition, Klenow fragment shows tighter binding to a mismatched G:G-B[a]P adducted primer-template than either a correctly base paired C:G-B[a]P or mismatched T:G-B[a]P primer template. The G:G-B[a]P structure also was inhibited from forming the closed ternary complex, yet dissociation rates from this complex were slower than for the correctly base paired C:G-B[a]P or mismatched T:G-B[a]P primer template. This indicates that the conformation adopted by the G:G-B[a]P within the active site of the polymerase is unique to this structure.
Federley, Richard George, "New insights into the mechanism of dna replication on unmodified and benzo[a]pyrene modified templates using surface plasmon resonance" (2011). Wayne State University Dissertations. 235.