![]() This polymerase also 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. 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). The DNA polymerase from bacteriophage T7 has been an important model system for understanding high-fidelity DNA replication due to its simple structure consisting of only two polypeptides (phage T7 gene product 5 and Escherichia coli thioredoxin) and its fast, accurate, and processive DNA replication. Phosphorothioate-modified DNAs have been subsequently employed in biotechnology to increase the stability of nucleic acids in vivo ( 14). Studies on phosphorothioate DNA supported this mechanism and afforded insights into the stereochemistry of the reaction ( 11, 12, 13). Subsequent crystal structures and kinetic studies on T4 DNA polymerase and Klenow Fragment polymerase identified conserved active site carboxylates that coordinate two metal ions at the exo site, suggesting a general two-metal ion mechanism ( 9, 10). Before structures were available, early work was complicated by the low sequence identity of different exonucleases as sequence alignment of exonuclease domains from different enzymes usually gives less than 15% sequence identity ( 8, 9). Early studies suggested that the substrate for the exonuclease reaction is at least partially single-stranded, rather than duplex DNA, based on the temperature dependence of the exonuclease reaction ( 7), but temperature dependence alone cannot distinguish alternative models because of expected differences in the thermodynamics of DNA binding at the pol (polymerase) and exo (exonuclease) sites. 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). High-fidelity polymerases also contain a 3′–5′ proofreading exonuclease that further increases replication fidelity by removing mismatches after they are incorporated. A large contribution to replication fidelity comes from a finely tuned polymerase active site that makes errors in only one out of 10 5 base pairs while incorporating nucleotides at rates exceeding 300 per second ( 1, 2). 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.ĭNA polymerases have evolved to efficiently copy genomes with extremely high fidelity to fulfill their critical role in maintaining genome stability. 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 show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. ![]() The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. ![]() 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. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 10 5, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. ![]()
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