TY - JOUR
T1 - Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase
AU - Dangerfield, Tyler L.
AU - Kirmizialtin, Serdal
AU - Johnson, Kenneth A.
N1 - Funding Information:
Funding and additional information—This work was supported by grants from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) R01AI110577 (to K. A. J) and National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) R01114223 (to K. A. J.). Computational research was carried out on the High-Performance Computing resources at New York University Abu Dhabi. S. K. is supported by AD181 Faculty Research grant. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2022 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2022/3/1
Y1 - 2022/3/1
N2 - 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 30-50 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 phosphorothioatemodified 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.
AB - 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 30-50 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 phosphorothioatemodified 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.
KW - DNA/chemistry
KW - DNA Replication
KW - DNA-Directed DNA Polymerase/chemistry
KW - Exonucleases
KW - Structure-Activity Relationship
KW - Substrate Specificity
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U2 - 10.1016/j.jbc.2022.101627
DO - 10.1016/j.jbc.2022.101627
M3 - Article
C2 - 35074426
AN - SCOPUS:85125011393
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 3
M1 - 101627
ER -