I. Introduction 61 II. Binding of Small Molecules to DNA 62 A. Covalent Binding 62 B. Reversible (Noncovalent) DNA-Binding Agents 65 III. DNA-Metal Ion Complexes 67 A. Platinum Complexes 70 B. Other Metal Ions 73 IV. DNA-Protein Complexes 74 A. Introduction 74 B. ESI-MS of DNA-Protein Complexes 76 C. ESI-MS Analysis of Proteolytic Products of DNA-Protein Complexes 79 D. ESI-MS of Ternary DNA-Protein-Ligand Complexes 80 V. Conclusions 80 Abbreviations 81 References 81 --Interactions of DNA with drugs, metal ions, and proteins are important in a wide variety of biological processes. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) is now a well-established tool for the characterization of the primary structures of biopolymers. The gentle nature of the ESI process, however, means that ESI-MS is also finding application for the study of noncovalent and other fragile biomolecular complexes. We outline here the progress, to date, in the use of ESI-MS for the study of noncovalent drug-DNA and protein-DNA complexes together with strategies that can be employed to examine the binding of small molecules and metal complexes to DNA. In the case of covalent complexes with DNA, sequence information can be derived from ESI-MS used in conjunction with tandem mass spectrometry (MS/MS) and/or enzymatic digestion. MS/MS can also be used to probe the relative binding affinities of drugs that bind to DNA via noncovalent interactions. Overall, the work in this area, to date has demonstrated that ESI-MS and MS/MS will prove to be valuable complements to other structural methods, offering advantages in terms of speed, specificity, and sensitivity. (c) 2001 John Wiley & Sons, Inc.

It has been hypothesized that G-quadruplexes can sequester the 3′ end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures implies that parallel telomeric G-quadruplexes may play an important biological role.

The structure of the glycerophosphodiesterase (GDPD) from Enterobacter aerogenes, GpdQ, has been solved by SAD phasing from the active site metal ions. Structural analysis indicates that GpdQ belongs to the alpha/beta sandwich metallo-phosphoesterase family, rather than the (alpha/beta)(8) barrel GDPD family, suggesting that GpdQ is a structurally novel GDPD. Hexameric GpdQ is generated by interactions between three dimers. The dimers are formed through domain swapping, stabilised by an inter-chain disulfide bond, and beta-sheet extension. The active site contains a binuclear metal centre, with a fully occupied alpha-metal ion site, and partially occupied beta-metal ion site, as revealed by anomalous scattering analysis. Using a combination of TLS refinement and normal mode analysis, the dynamic movement of GpdQ was investigated. This analysis suggests that the hexameric quaternary structure stabilises the base of the dimer, which promotes "breathing" of the active site cleft. Comparison with other metallo-phosphodiesterases shows that although the central, catalytic, domain is highly conserved, many of these enzymes possess structurally unrelated secondary domains located at the entrance of the active site. We suggest that this could be a common structural feature of metallo-phosphodiesterases that constrains substrate specificity, preventing non-specific phosphodiester hydrolysis.

Processive DNA synthesis by the aeh core of the Escherichia coli Pol III replicase requires it to be bound to the b 2 clamp via a site in the a polymerase subunit. How the e proofreading exonuclease subunit influences DNA synthesis by a was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of e. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel b-binding site in e that, in conjunction with the site in a, maintains a closed state of the aeh-b 2 replicase in the polymerization mode of DNA synthesis. The e-b interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

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The core of DNA polymerase III, the replicative polymerase in Escherichia coli, consists of three subunits (alpha, epsilon, and theta). The epsilon subunit is the 3'-5' proofreading exonuclease that associates with the polymerase (alpha) through its C-terminal region and theta through a 185-residue N-terminal domain (epsilon 186). A spectrophotometric assay for measurement of epsilon activity is described. Proteins epsilon and epsilon 186 and the epsilon 186.theta complex catalyzed the hydrolysis of the 5'-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of k(cat) and K(M), confirming that the N-terminal domain of epsilon bears the exonuclease active site, and showing that association with theta has little direct effect on the chemistry occurring at the active site of epsilon. On the other hand, formation of the complex with theta stabilized epsilon 186 by approximately 14 degrees C against thermal inactivation. For epsilon 186, k(cat) = 293 min(-)(1) and K(M) = 1.08 mM at pH 8.00 and 25 degrees C, with a Mn(2+) concentration of 1 mM. Hydrolysis of pNP-TMP by epsilon 186 depended absolutely on divalent metal ions, and was inhibited by the product TMP. Dependencies on Mn(2+) and Mg(2+) concentrations were examined, giving a K(Mn) of 0.31 mM and a k(cat) of 334 min(-1) for Mn(2+) and a K(Mg) of 6.9 mM and a k(cat) of 19.9 min(-1) for Mg(2+). Inhibition by TMP was formally competitive [K(i) = 4.3 microM (with a Mn(2+) concentration of 1 mM)]. The pH dependence of pNP-TMP hydrolysis by epsilon 186, in the pH range of 6.5-9.0, was found to be simple. K(M) was essentially invariant between pH 6.5 and 8.5, while k(cat) depended on titration of a single group with a pK(a) of 7.7, approaching limiting values of 50 min(-1) at pH <6.5 and 400 min(-1) at pH >9.0. These data are used in conjunction with crystal structures of the complex of epsilon 186 with TMP and two Mn(II) ions bound at the active site to develop insights into the mechanisms of pNP-TMP hydrolysis by epsilon at high and low pH values.

Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp.

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO 2 -fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob AtL plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L 8 A S 8 t Rubisco [comprising AtL and tobacco small (S) subunits] in tob AtL-R1 leaves compared with tob AtL , despite >threefold lower steady-state Rubisco mRNA levels in tob . Accompanying twofold increases in photosynthetic CO 2 -assimilation rate and plant growth were measured for tob lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.T he increasing global demands for food supply, bioenergy production, and CO 2 -sequestration have placed a high need on improving agriculture yields and resource use (1, 2). It is now widely recognized that yield increases are possible by enhancing the light harvesting and CO 2 -fixation processes of photosynthesis (3-5). A major target for improvement is the enzyme Rubisco [ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase] whose deficiencies in CO 2 -fixing speed and efficiency pose a key limitation to photosynthetic CO 2 capture (6, 7). In plants, the complex, multistep catalytic mechanism of Rubisco to bind its 5-carbon substrate RuBP, orient its C-2 for carboxylation, and then process the 6-carbon product into two 3-phosphoglycerate (3PGA) products, limits its throughput to one to four catalytic cycles per second (8). The mechanism also makes Rubisco prone to competitive inhibition by O 2 that produces only one 3PGA and 2-phosphoglycolate (2PG). Metabolic recycling of 2PG by photorespiration requires energy and results in most plants losing 30% of their fixed CO 2 (5). To compensate for these catalytic limitations, plants like rice and wheat invest up to 50% of the leaf protein into Rubisco, which accounts for ∼25% of their leaf nitrogen (9).Natural diversity in Rubisco catalysis demonstrates that plant Rubisco is not the pinnacle of evolution (6, 7). Better-performing versions in some red algae have the potential to raise the yield of ...