Abstract
High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension with the correct nucleotide, the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. The simulations suggest that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, nonideal base pairing, and a large increase in the distance from the 3ʹ-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high-fidelity DNA polymerases.
Highlights
The molecular basis for the extraordinary specificity of enzymes has been a longstanding question in enzymology
DNA polymerases provide an optimal system for understanding enzyme specificity because fidelity and speed of DNA replication are biologically important for maintaining genome integrity, the alternate substrates are well defined, and crystal structures show large conformational changes in the enzyme after nucleotide binding [3]
As an alternative approach to using dideoxy-terminated oligonucleotides, we attempted to use the nonhydrolyzable nucleoside analog dTpNpp with an oligonucleotide substrate containing a normal 3ʹ-OH group in the primer strand to measure nucleotide binding kinetics; we found that this analog was a very poor substrate for T7 DNA polymerase
Summary
While kcat/Km values for a correct- and a mismatchednucleotide incorporation have been reported [8, 29,30,31,32,33], general questions about misincorporation still remain, especially for high-fidelity DNA polymerases. To fit the chemical-quench data along with the stopped-flow data, the model required an additional step where dTTP binds to the FDT state and activates the enzyme (forming the GDT0 state) to stimulate the rate of product formation (Fig. 5). The 3ʹ-OH group of the primer strand is misaligned, with an average distance from the α-phosphate of the incoming nucleotide of 5.7 ± 0.3 Å While mismatched nucleotide binding is weakened, larger effects can be seen on the alignment of catalytic residues and alignment of the 3ʹ-OH, which is known to contribute to the greatly reduced rate of chemistry
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
