Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution

Abstract

The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3 -hydroxyl of the growing DNA chain. Here we report a singlemolecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Fö rster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.Klenow Fragment ͉ polymerase and exonuclease site ͉ single molecule fluorescence ͉ single nucleotide resolution ͉ structural dynamics T he catalytic mechanism of Escherichia coli DNA polymerase I has been rigorously studied for more than 40 years (1, 2). The E. coli DNA polymerase I (Klenow fragment [KF]), an active truncated form of polymerase I, is composed of two domains: a polymerase domain that incorporates nucleotides, and a 3Ј-5Ј exonuclease domain that excises misincorporated nucleotides. The polymerase domain consists of three subdomains: the fingers, the palm, and the thumb. The fingers subdomain is primarily involved in interactions with the singlestranded region of the DNA template and the incoming nucleotide; the palm forms the active site of the polymerase upon interaction with the incoming dNTP; and the thumb is responsible for binding double-stranded DNA. The exonuclease domain, located Ϸ30 Å from the polymerase domain, binds to the 3Ј-terminus of the primer when a mismatched base is incorporated (3).Like other high-fidelity polymerases, KF achieves its extraordinary accuracy through a series of steps that discriminate between a correct and incorrect dNTP. A minimal reaction pathway for KF has been proposed with much of the data obtained from chemical quench experiments (4, 5) (Fig. 1A). The rate-limiting step (k 3 ) that precedes the phosphoryl-transfer step (k 4 ) had been tentatively attributed to a conformational change of the fingers domain (6). A comparison of the crystal structures of the binary polymerase-DNA complexes with those of the ternary polymerase-DNA-dNTP complexes reveals a substantial movement upon nucleotide binding, supporting the model that fingers closing was the rate-limiting step (7). However, recent results have shown this step is much too fast to be rate limiting, suggesting additional noncovalent steps th...

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

“…We [and others (17,20,21)] have observed that under most conditions the primer strand at P/T DNA junctions populates both the pol and exo sites of binary DNAP complexes, even in the absence of a terminal mismatch. DNA binding in the exo site is usually thought to be involved in the replication pathway only after covalent misincorporation; that is, in the context of primer editing to remove an incorrect 3′ terminal base.…”

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

“…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).…”

Replication infidelity via a mismatch with Watson–Crick geometry

“…While all DNA polymerases studied to date can occasionally incorporate an incorrect nucleotide, misincorporation is usually rare. This is because most DNA polymerases employ a series of prechemistry conformational transitions as checkpoints for exclusion of incorrect substrates (9)(10)(11). For this reason it is difficult to obtain structural support for Watson and Crick's hypothesis on the origin of spontaneous base substitutions.…”