Structure-specific DNA-induced Conformational Changes in Taq Polymerase Revealed by Small Angle Neutron Scattering

Abstract

The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5 -nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNAbound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.In addition to its well known in vitro applications in PCR, in vivo the DNA polymerase I from Thermus aquaticus (Taq polymerase) performs nucleotide replacement reactions in DNA repair and RNA primer removal in Okazaki fragment processing during lagging-strand synthesis (1). Taq polymerase also utilizes both its polymerase and 5Ј-nuclease activities in a nucleotide replacement reaction: it catalyzes the upstream addition of deoxynucleoside 5Ј-triphosphates to the 3Ј-hydroxyl terminus of an RNA primer and also cleaves the downstream, single-stranded 5Ј-nucleotide displaced by the growing upstream strand. On a DNA duplex substrate, upstream primer extension and downstream strand excision must be highly coordinated (2) to leave a ligatable nick, instead of a gap or an overhang, on a DNA duplex that is to be sealed later by a DNA ligase.The structure and functional properties of Taq polymerase are similar to those of the polymerase I of Escherichia coli (3). Taq polymerase has two distinct domains: an N-terminal domain (residues 1-289) that has single-stranded 5Ј-nucleotide cleavage activity (5Ј-nuclease domain), and a C-terminal domain (residues 306 -832) that has DNA polymerase activity (Klentaq domain). Integrated into the Klentaq domain is another domain (residues 306 -423) that is structurally analogous to the proof editing 3Ј 3 5Ј-exonuclease domain of E. coli polymerase I, but this domain has n...

Coupled protein domain motion inTaqpolymerase revealed by neutron spin-echo spectroscopy

“…The first six normal modes are the translational and rotational motions of Taq polymerase as a whole (not shown). The lowest internal seventh and eighth normal modes are relative en bloc motions between Klentaq and 5Ј nuclease with 5Ј nuclease moving toward the thumb, which is functionally important (17). Subdomain motions become apparent for the 9th, 10th, and higher normal modes.…”

“…During such processes, Taq polymerase utilizes a DNA polymerase domain to catalyze the addition of dNTP to the 3Ј hydroxyl terminus of an RNA primer and a 5Ј nuclease domain to cleave the downstream, single-stranded 5Ј nucleotide displaced by the growing upstream strand (11). Because the structure of Taq polymerase possesses an extended conformation with the polymerase and the 5Ј nuclease active sites separated by Ϸ70 Å (15)(16)(17), the DNA needs to be shuttled between these two distant catalytic sites when switching from the DNA synthesis mode to the nucleotide cleavage mode. This scenario is similar to that which occurs when the DNA needs to be shifted from the polymerase active site to the 3Ј-5Ј exonuclease catalytic center, which are Ϸ30 Å apart in the Klenow fragment (the polymerase domain plus the 3Ј-5Ј exonuclease domain) domain of polymerase I, to cleave an incorrectly incorporated dNTP (18).…”

“…Here, we show that NSE can resolve the domain mobility tensor of a protein, thus specifying how protein domains are dynamically coupled during global conformational changes. (17). NSE experiments were conducted at the Institut für Festkör-perforschung (27).…”

Ezrin Controls the Macromolecular Complexes Formed between an Adapter Protein Na+/H+ Exchanger Regulatory Factor and the Cystic Fibrosis Transmembrane Conductance Regulator

“…The program Situs (47) was used to dock the fragments of known crystal structures (PDZ1 and ezFERM) to the low resolution structures. More details about the procedures for reconstruction of the three-dimensional molecular images from solution SAXS data have been described previously (48).…”

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

“…At the contrastmatching point of the PIP 2 lipid, SANS determines the conformational changes of the deuterated proteins that have sufficient coherent neutron scattering without the interference scattering from the lipid. The concept and applications of contrastmatching small angle scattering have been described elsewhere (27,50,51,60,62,67,68). (56).…”