Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Abstract

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles

“…A caveat of these force‐based experiments is that polymerase and exonuclease activities are measured by applying tension to the primer–template junction that stretches the template DNA and creates a scenario equivalent to an incorrectly incorporated nucleotide. This is consistent with the observation that the probability of primer‐end binding to the exonuclease site was proportional to the tension (Hoekstra et al , ). Moreover, the constant tension on the template created a scenario akin to a continuous series of misincorporated nucleotides, which explains why the primer‐end remains at the Exo‐site for an extended period, and there is processive excision of a large number of nucleotides.…”

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

“…Without exogenous PPi, Pfu polymerase can excise either a single terminal nucleotide or eight nucleotides at the 3′ end. To proceed with exonucleolytic cleavage, the 3′ end of the hairpin has to originally be bound at or translocated into exonuclease domain 43 – 45 . In this case, Pfu polymerase generates partially degraded hairpin along with H4-dT.…”

Accurate flexible refinement of atomic models against medium-resolution cryo-EM maps using damped dynamics

“…The original “Damped-Dynamics Flexible Fitting” (DDFF) simulation engine [ 5 ], on which we base our present development, predates the revolutionary improvement in cryo-EM techniques, and was targeted mainly to low-resolution EM maps. Several new flexible-fitting approaches have been published since then, which can be broadly grouped into a few types: Methods based on elastic network models [ 6 – 10 ]. Methods based on molecular-dynamics simulations [ 11 – 14 ].…”

“… Methods based on graph-theoretic approaches to determine correspondences between secondary-structure elements [ 15 – 17 ]. Others: Force field heuristically defined in terms of the “overlap” between the EM map and the model [ 18 ]; Monte-Carlo optimization of fragments combined with all-atom refinement [ 19 ]; Bayesian-based refinement [ 20 ]. …”