Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site.

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.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

We have used site‐directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3′ terminus bound at the 3′‐5′ exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3′ terminus. The pH‐dependence of the 3′‐5′ exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme‐bound metal ions.

Toward the goal of identifying complete sets of transcription factor (TF)-binding sites in the genomes of several gamma proteobacteria, and hence describing their transcription regulatory networks, we present a phylogenetic footprinting method for identifying these sites. Probable transcription regulatory sites upstream of Escherichia coli genes were identified by cross-species comparison using an extended Gibbs sampling algorithm. Close examination of a study set of 184 genes with documented transcription regulatory sites revealed that when orthologous data were available from at least two other gamma proteobacterial species, 81% of our predictions corresponded with the documented sites, and 67% corresponded when data from only one other species were available. That the remaining predictions included bona fide TF-binding sites was proven by affinity purification of a putative transcription factor (YijC) bound to such a site upstream of the fabA gene. Predicted regulatory sites for 2097 E.coli genes are available at http://www.wadsworth.org/resnres/bioinfo/.

The 440-amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168-residue mini-intein, ΔI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (ΔI-CM) increased C-terminal cleavage activity. Using the I-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139-residue mini-intein, ΔΔI hh -SM, by replacing 36 amino acids in the residual endonuclease loop with a seven-residue β-turn from the autoprocessing domain of Hedgehog protein. The threedimensional structures of ΔI-SM, ΔΔI hh -SM, and two variants, ΔΔI hh -CM and ΔΔI hh , have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N-and C-termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side chain of Asp422 plays a role in both N-and C-terminal cleavage, thereby suggesting that this highly-conserved residue regulates the balance between the two reactions.

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.

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