Abstract
Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations.
Highlights
DNA polymerases have evolved to efficiently copy genomes with extremely high fidelity to fulfill their critical role in maintaining genome stability
We address the temperature dependence of the proofreading reaction, measure the kinetics of extension versus excision of buried mismatches, and we propose a structural model for the exonuclease active site based on homology modeling refined by molecular dynamics (MD) simulation
We performed our previous kinetic studies on the correct nucleotide incorporation for T7 DNA polymerase at 4°C in order to better resolve the fast rate of the conformational change step, so we initially performed experiments to characterize the exonuclease activity at 4°C to be consistent with the previous results [1]
Summary
DNA polymerases have evolved to efficiently copy genomes with extremely high fidelity to fulfill their critical role in maintaining genome stability. High fidelity polymerases contain a 3’–5’ proofreading exonuclease that further increases replication fidelity by removing mismatches after they are incorporated The contribution of this activity to fidelity varies depending on the enzyme [3], ranging from a factor of ~10 to more than 10,000 [4,5,6]. We previously determined the complete kinetic pathway of correct nucleotide incorporation and misincorporation by T7 DNA polymerase using a variant with a fluorescent artificial amino acid [15,16] This polymerase features a 3’–5’ proofreading exonuclease active site within the N-terminal region of gene product 5, approximately 35 Å from the polymerase active site. Our data demonstrate that the fidelity of this enzyme may be much higher than previously estimated and shows key active site differences when compared with other DNA polymerases with proofreading exonuclease domains
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