Abstract
DNA replication occurs in vivo with very high processivity, meaning that the replication complex assembles at the origin(s) of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation. Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro. However, the isolated DNA polymerases are much less processive, especially under physiological conditions. In this paper we monitor the degree of processivity displayed by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can "edit-out" the 3'-terminal nucleotide from the primer (using the 3'----5'-exonuclease activity of the polymerase) and then switch into the synthesis mode without dissociating from the DNA template. This "switch experiment" is accomplished by using mismatched primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation event has occurred. By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution. Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully processive manner. We also show that the removal of a doubly mispaired sequence from the 3'-terminus of the primer, followed by synthesis, is comparably processive. In contrast, the apparent processivity of removing a triply mispaired terminus is much reduced. Taken together, these observations are consistent with the notion that the "editing active site" of the T4 enzyme optimally accommodates only two unpaired nucleotide residues. Our results do not support the idea that the exonuclease activity of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated base determines overall editing efficiency. The integrated results of this study provide additional insight into the structure of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that are required to provide the holoenzyme complex with full processivity.
Highlights
DNA replication occurs in vivowith veryhigh proc- DNA replication complexes involved in the elongation essivity, meaning that thereplication complex assem- phase of replication in prokaryotes and eukaryotes contain a bles a t the origin(s) of replication and performs number of subunits, ranging from two for the simplest Eschtemplate-directed synthesis of DNA over virtually the erichia coli bacteriophage (T7) toover 20 for the entire funcentire genome without dissociation
Aside from the obvious logistical problems associated with reassembling the complex at each nucleotide insertion step, simple calculations for the T4DNA replication system have shown that the time that would be required for that the dwell time at a misincorporated base deter- diffusion to bring just thepolymerase itself back to theprimermines overall editingefficiency
We have investigated the processive editing by T4 DNA polymerase of various P/T constructs using a single-turnover protocol
Summary
Use of Heparin to Obtain Single-turnoverConditions-The processivity of T4 DNA polymerase or holoenzyme has previously been measured in our laboratory using “single-hit” (Newport et al.,1980; Dolejsi, 1988J;arvis et al, 1990a, 1991) as well as “single-turnover” (Jarvis et al.,1990a, 1991)kinetic conditions. We have investigated the processive editing by T4 DNA polymerase of various P/T constructs using a single-turnover protocol This condition is attained by the strategic use of a reagent to “trap” the polymerase molecules that dissociate from the P/T substrateaftera reaction has been initiated, thereby preventing polymerase rebinding and “multiple-hit” extensionof the DNA primer. We have monitored the processivity of the 3’4’-exonucleaseactivity of T4 DNA polymerase on P/ T substrates Products reflecting this exonuclease activity in a multihit (untrapped) regime are obtained when the polymerase is preincubated with (e.g.1 the 17/25 P/T construct and the reaction is initiated by the addition of M$+ alone ( l a n e 2, Fig. 1).Here the T4 DNA polymerase effectively digests the fully base-paired primer strand by removing at least nucleotide residues in less than s. A dramatically different result is obtained when the same reaction is initiated by the simultaneous addition of
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