Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

Abstract

The crystal structure ofthe large fragment of the Thermus aquaticus DNA polymerase (Klentaql), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaql differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaql reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.Amplification of DNA fragments by the polymerase chain reaction (PCR) has become an important and widespread tool of genetic analysis since the introduction of the thermostable DNA polymerase from Thermus aquaticus (Taq) (1-3). The enzyme, by enabling the amplification reaction to be performed at higher temperatures, allows the convenience of heat denaturation of DNA without enzyme inactivation. Purified Taq DNA polymerase, however, is devoid of 3'-5' exonuclease activity and thus cannot excise misincorporated nucleotides (4, 5). Consequently, DNA amplification by the Taq DNA polymerase is an error-prone process. Enzymes with N-terminal deletions show a reduced tendency toward errors, as do some recently discovered thermostable DNA polymerases which have an integral editing exonuclease activity (6, 7). The latter enzymes, however, are unable to amplify sequences in excess of 5.0 to 7.0 kb that full-length Taq DNA polymerase (8) or N-terminally deleted enzyme (7) can amplify readily. The amplification of very large I)NA fragments (up to 35 kb) was recently achieved by combining an N-terminally deleted Taq DNA polymerase called Klentaql with a low level of an archaebacterial thermostable DNA polymerase exhibiting 3'-5' exonuclease activity (9, 10). Taq DNA polymerase or forms of the enzyme with N-terminal deletions are also used in DNA sequencing (10-12). However, the quality of the data has been limited and the expense kept high by the poor affinity of the enzyme for dideoxynucleotides. Mutants with increased affinity for chain terminators would be of considerable interest.To understand the structural basis of thermostability and provide the foundation for the improvement of the Taq DNA polymerase, we present here the three-dimensional structure of Klentaql.t extension (MGKRKST) was used and yielded crystals diffracting to beyond 2.5-A resolution (Table 1). Crystals of Klentaql were obtained at room temperature by using vapor diffusion against a solution of 6% (wt/vol) polyethylene glycol 3350/50 mM MgCl2/100 mM Tris-HCl, pH 9.0, starting with equal mixtures of protein and polyethylene glycol solutions (13). Klentaql crystals are in space group P21212 (a = 109.4 A, b = 136.8 A, c = 45.6 A) with one molecule in the asymmetric unit.Structure Determination. Details of structure determination will be published elsewhere. In brief, heavy-atom derivatives were pr...

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

“…2B). The observed FRET efficiency (Ϸ0.68) corresponds to a distance of Ϸ53 Å (R 0 ϭ 60 Å) (13), which is within 3 Å of the estimated distance from the Klentaq crystal structure (Ϸ50 Å) (14). The dwell-time distributions (Fig.…”

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…S4). In KlenTaq 1QTM ddTTP was bound to the active site due to the applied crystallization strategy (24)(25)(26)28). A zoom into the active site shows that the dT spin TP is located opposite to adenine of the template strand, forming Watson-Crick base pairing interactions to the templating base ( Fig.…”

“…To reveal the structural basis for the ability of DNA polymerases to process an artificial substrate we crystallized KlenTaq as ternary complex bound to a primer/template duplex and a respective C5 modified triphosphate in the incoming position. The crystals were obtained by a strategy previously used for KlenTaq bound to unmodified substrates (24)(25)(26)28). By incorporation of ddCMP at the primer terminus a primer/template duplex lacking a 3′-terminal hydroxyl group is formed to terminate the primer extension reaction.…”

“…The large fragment of Thermus aquaticus (Taq) DNA polymerase (in short KlenTaq, N-terminally truncated form of Taq polymerase) was chosen as the target because this enzyme class is heavily employed and well characterized on a functional and structural level (24)(25)(26)(27)(28)(29)(30)(31). We compared two dNTP analogsnamely dT spin TP and dT dend TP (Fig.…”

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

“…It is a member of the family A DNA polymerase group (8,9), of which Escherichia coli pol I is the prototype (10). The crystal structures of numerous family A DNA polymerases have been solved, revealing a high degree of structural similarity among its members (11)(12)(13)(14)(15)(16)(17). The interaction between DNA polymerase and template-primer DNA has also been revealed in molecular detail by structural determination of pol:DNA co-crystals and in biochemical studies.…”