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A K Showalter

Publications and source records attributed to A K Showalter.

4 recordsLinked to original sources
PubMed2001

Insight into the catalytic mechanism of DNA polymerase beta: structures of intermediate complexes.

The catalytic reaction mediated by DNA polymerases is known to require two Mg(II) ions, one associated with dNTP binding and the other involved in metal ion catalysis of the chemical step. Here we report a functional intermediate structure of a DNA polymerase with only one metal ion bound, the DNA polymerase beta-DNA template-primer-chromium(III).2'-deoxythymidine 5'-beta,gamma-methylenetriphosphate [Cr(III).dTMPPCP] complex, at 2.6 A resolution. The complex is distinct from the structures of other polymerase-DNA-ddNTP complexes in that the 3'-terminus of the primer has a free hydroxyl group. Hence, this structure represents a fully functional intermediate state. Support for this contention is provided by the observation of turnover in biochemical assays of crystallized protein as well as from the determination that soaking Pol beta crystals with Mn(II) ions leads to formation of the product complex, Pol beta-DNA-Cr(III).PCP, whose structure is also reported. An important feature of both structures is that the fingers subdomain is closed, similar to structures of other ternary complexes in which both metal ion sites are occupied. These results suggest that closing of the fingers subdomain is induced specifically by binding of the metal-dNTP complex prior to binding of the catalytic Mg(2+) ion. This has led us to reevaluate our previous evidence regarding the existence of a rate-limiting conformational change in Pol beta's reaction pathway. The results of stopped-flow studies suggest that there is no detectable rate-limiting conformational change step.

Animals↗
PubMed2001

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.

African Swine Fever Virus↗
PubMed2000

DNA polymerase beta: contributions of template-positioning and dNTP triphosphate-binding residues to catalysis and fidelity.

The specific catalytic roles of two groups of DNA polymerase beta active site residues identified from crystal structures were investigated: residues possibly involved in DNA template positioning (Lys280, Asn294, and Glu295) and residues possibly involved in binding the triphosphate moiety of the incoming dNTP (Arg149, Ser180, Arg183, and Ser188). Eight site-specific mutants were constructed: K280A, N294A, N294Q, E295A, R149A, S180A, R183A, and S188A. Two-dimensional NMR analysis was employed to show that the global conformation of the mutants has not been perturbed significantly. Pre-steady-state kinetic analyses with single-nucleotide gapped DNA substrates were then performed to obtain the rate of catalysis at saturating dNTP (k(pol)), the apparent dissociation constant for dNTP (K(d)), catalytic efficiency k(pol)/K(d), and fidelity. Of the three template-positioning residues, Asn294 and Glu295 (but not Lys280) contribute significantly to k(pol). Taken together with other data, the results suggest that these two residues help to stabilize the transition state during catalysis even though they interact with the DNA template backbone rather than directly with the incoming dNTP or the opposite base on the template. Furthermore, the fidelity increases by up to 19-fold for N294Q due to differential k(pol) effects between correct and incorrect nucleotides. Of the four potential triphosphate-binding residues, Ser180 and Arg183 contribute significantly to k(pol) while the effects of R149A are relatively small and are primarily on K(d), and Ser188 appears to play a minimal role in the catalysis by Pol beta. These results identify several residues important for catalysis and quantitate the contributions of each of those residues. The functional data are discussed in relation to the prediction on the basis of available crystal structures.

Animals↗