High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting ofthe duplex DNA by the protein is stabilized by hydophobic interactions between Phe473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced.The large proteolytic fragment (Klenow fragment) of Escherichia coli DNA polymerase I utilizes an editing 3'-5' exonuclease activity (1) to reduce the misincorporation of erroneous nucleotides by about 10-fold (2) at an active site that is some 30 A away from the polymerase site of misincorporation (3). How might this be accomplished? The crystal structure of the Klenow fragment shows that it is folded into two domains (3). Various experiments (reviewed in ref. 4) establish that the domain to which the dNMP binds in the crystal catalyzes the 3'-5' econuclease activity, whereas the larger C-terminal domain contains the active site for the polymerase reaction. Mutant proteins that contain amino acid changes in the dNMP binding site have been made by directed mutagenesis; they are devoid of exonuclease activity but retain full polymerase activity (5). Furthermore, the DNA encoding the C-terminal domain has been cloned, and the product has been expressed, isolated, and shown to possess significant DNA polymerase activity with no measurable 3'-5' exonuclease activity (6). The observation (3) that these two active sites are -25-30 A apart poses the interesting question of how they work together to achieve high-fidelity synthesis of DNA.The C-terminal domain contains a cleft that is large enough to accommodate the double-stranded B-DNA product of DNA synthesis (3). The approximate position of the 3' terminus of the primer strand has been derived from the cross-linking of 8-azido-dATP to Tyr-776, footprinting of Klenow fragment on DNA (7), and the position of sitedirected mutants that alter polymerase activity but not exonuclease activity (A. Polesky and C. Joyce, personal communication). This model of DNA at the polymerase active site places about 8 base pairs (bp) of duplex product DNA in the cleft.A more detailed understanding of the structural basis ofthe polymerase and exonuclease activities requires the separate determination of the crystal structures of suitable DNAs complexed with each of these ...

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

This paper presents an investigation into the utility of document summarisation in the context of information retrieval, more specifically in the application of so called query biased (or user directed) summaries: summaries customised to reflect the information need expressed in a query. Employed in the retrieved document list displayed after a retrieval took place, the summaries' utility was evaluated in a task-based environment by measuring users' speed and accuracy in identifying relevant documents. This was compared to the performance achieved when users were presented with the more typical output of an IR system: a static predefined summary composed of the title and first few sentences of retrieved documents. The results from the evaluation indicate that the use of query biased summaries significantly improves both the accuracy and speed of user relevance judgements.

Type II topoisomerases alter DNA topology by forming a covalent DNA-cleavage complex that allows DNA transport through a double-stranded DNA break. We present the structures of cleavage complexes formed by the Streptococcus pneumoniae ParC breakage-reunion and ParE TOPRIM domains of topoisomerase IV stabilized by moxifloxacin and clinafloxacin, two antipneumococcal fluoroquinolones. These structures reveal two drug molecules intercalated at the highly bent DNA gate and help explain antibacterial quinolone action and resistance.

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg2+ ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.

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Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.