DNA conformational changes at the primer-template junction regulate the fidelity of replication by DNA polymerase

Abstract

Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3′-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3′-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.DNA editing | Klenow DNA polymerase | primer partitioning in DNAP active sites | low energy circular dichroism | base analogues 6-MI and 2-AP

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

“…Yet, recent magnetic-tweezers studies of T4 and T7 DNAp showed switching only from exo to pol (12). With our approach we can infer a direct switch in enzyme activity from a sudden change in the direction of the trace (see Fig.…”

“…The decrease of the polymerization rate can be explained by T7 DNAp undergoing force-sensitive structural changes during polymerization, when the finger domains have to close to correctly align and incorporate a nucleotide (22). In a physiological setting, our high-tension situation could correspond to an erroneous nucleotide that cannot properly basepair with the template strand, increasing both the probability of unbinding of the incoming nucleotide and the probability of unbinding of DNAp from its pol active site and rebinding with the exo active site (12,23,24). We find that the probability of entering an incorporation pause increases fivefold when the tension is increased to >35 pN.…”

“…It also has been shown that the apparent rate of polymerization goes down as tension increases up to~35 pN (8), after which DNAp seems to exclusively perform exonucleolysis (10,11). In a single-molecule Förster resonance energy transfer study using fluorescent base analogs, Klenow polymerase was shown to bind relaxed, matched PTS with both its pol and exo active sites (12,13). Moreover, the replicative T4 and T7 DNAp (families B and A, respectively) were previously shown to exhibit similar behavior in preferentially performing exonucleolysis when polymerization was obstructed by dsDNA ahead of the DNAp (14).…”

Distinct Complexes of DNA Polymerase I (Klenow Fragment) for Base and Sugar Discrimination during Nucleotide Substrate Selection

“…Perhaps this movement occurs when Tyr-766, which occupies the templating site in crystal structures of A family DNA polymerase binary complexes, is displaced by the N ϭ 0 base that occupies this position in crystal structures of polymerase-DNA-dNTP ternary complexes (26 -28). Finally, it is possible the movement we detect as an amplitude shift corresponds to a recently reported change in circular dichroism spectra that occurs in response to complementary dNTP binding to KF-DNA complexes and that is detected using probes at template positions N ϭ 0 and N ϭ ϩ1 (29).…”

Fifty years of DNA “Breathing”: Reflections on old and new approaches

“…For example, we were able to use limited nucleotide sets to ‘walk’ DNA or RNA polymerases from one defined position in a nucleic acid construct to another, and thus determine the spectral changes that occurred as these proteins moved into and through known DNA or RNA terminators, pause sites, hairpins, etc. 42 , 43 , 46 . By using, in particular, the 2-dimensional fluorescence spectra (2DFS) approach described in Section V below, we have recently been able to start to realize the ‘spectroscopist’s dream’ of making actual structural interpretations of such spectra.…”