Nigel D. F. Grindley

The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

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.

Conformational transitions in DNA polymerase I revealed by single-molecule FRET

The remarkable fidelity of most DNA polymerases depends on a series of early steps in the reaction pathway which allow the selection of the correct nucleotide substrate, while excluding all incorrect ones, before the enzyme is committed to the chemical step of nucleotide incorporation. The conformational transitions that are involved in these early steps are detectable with a variety of fluorescence assays and include the fingers-closing transition that has been characterized in structural studies. Using DNA polymerase I (Klenow fragment) labeled with both donor and acceptor fluorophores, we have employed single-molecule fluorescence resonance energy transfer to study the polymerase conformational transitions that precede nucleotide addition. Our experiments clearly distinguish the open and closed conformations that predominate in Pol-DNA and Pol-DNA-dNTP complexes, respectively. By contrast, the unliganded polymerase shows a broad distribution of FRET values, indicating a high degree of conformational flexibility in the protein in the absence of its substrates; such flexibility was not anticipated on the basis of the available crystallographic structures. Real-time observation of conformational dynamics showed that most of the unliganded polymerase molecules sample the open and closed conformations in the millisecond timescale. Ternary complexes formed in the presence of mismatched dNTPs or complementary ribonucleotides show unique FRET species, which we suggest are relevant to kinetic checkpoints that discriminate against these incorrect substrates.

IS1 insertion generates duplication of a nine base pair sequence at its target site

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.

Transposon-mediated site-specific recombination: Identification of three binding sites for resolvase at the res sites of γδ and Tn 3

Abstract The tnpR gene product, resolvase, of the transposable element γδ mediates site-specific recombination between two copies of γδ directly repeated on the same replicon, and it negatively regulates transcription of the tnpA (transposase) gene and its own gene. The recombinational site, res, and the regulatory region both are located in the tnpA-tnpR intercistronic region. In studying the interaction of purified resolvase with DNA fragments derived from γδ and the related transposon, Tn 3, that span this region, we have demonstrated that three sites specifically bind resolvase. Site I overlaps the recombinational crossover point and both transcriptional promoters. Sites II and III cover most of the DNA between the crossover point and the translational start of the tnpR gene. These are the only binding sites we have detected in a region of about 400 base pairs centered on the crossover point. Studies of cointegrates that contain only part of the region that binds resolvase indicate that site I is not sufficient for efficient site-specific recombination and suggest that all three sites are probably required.

Fingers-Closing and Other Rapid Conformational Changes in DNA Polymerase I (Klenow Fragment) and Their Role in Nucleotide Selectivity†

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca (2+) instead of Mg (2+) as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

The crystal structure of the catalytic domain of the site-specific recombination enzyme γδ resolvase at 2.7 Å resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.

DNA transposition: From a black box to a color monitor

It is nearly half a century since the classical genetic experiments of Barbara McClintock revealed to her the existence of transposing DNA elements in maize (see McClintock, 1992). The intervening years have witnessed the identification of an ever-increasing number of similar elements from the lowly bacterial insertion sequences to the complex genome of bacteriophage Mu and from the invaluable P element, tool of Drosophila genetics, to the medically devastating HIV retrovirus. Time has also seen dramatic growth in our level of knowledge and understanding of DNA transposition as the geneticists have given way first to the molecular biologists, then to the biochemists, and now to the X-ray crystallographers. In four recent papers, the crystal structures of the catalytic domains of three transposase proteins have been reported, starting in December of last year with the HIV integrase and now with the Mu transposase and the avian sarcoma virus (ASV) integrase (Dyda et al., 1994; Rice and Mizuuchi, 1995; Bujacz et al., 1995, 1996).

Cooperativity mutants of the γδ resolvase identify an essential interdimer interaction

gamma delta resolvase, a transposon-encoded site-specific recombinase, catalyzes the resolution of the cointegrate intermediate of gamma delta transposition. The recombination reaction involves the formation of a catalytic nucleoprotein complex whose structure is determined by specific protein-DNA and protein-protein interactions. We have isolated many resolvase mutants and have identified four that are unable to mediate a subclass of higher order protein-protein interactions necessary for recombination. This mutant phenotype is characterized by an inability to catalyze recombination, a loss of cooperative binding to res DNA, and a failure to induce looping out of the DNA between two resolvase binding sites within res. The amino acid side chains identified by the cooperativity mutants cluster on a surface of the protein that mediates an interaction between resolvase dimers in a crystallographic tetramer. We have therefore identified a region of resolvase that mediates an interdimer protein-protein interaction necessary for the formation of the recombinogenic synaptic intermediate.

The γδ resolvase induces an unusual DNA structure at the recombinational crossover point

gamma delta resolvase, a transposon-encoded protein active in site-specific recombination, induces a structural change in the DNA at the recombinational crossover point that results in enhanced intercalation of the foot-printing reagent MPE X Fe(II). The structural change correlates with the formation of a bend in the DNA: a mutant resolvase that binds to the crossover site but induces little or no bend does not promote intercalation. The properties of the mutant protein suggest that the induced structural change, which we propose is a localized kink, is required for recombination.

Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

The Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed −1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure‐eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single‐stranded version of the minicircle junction — the precise 3′ end of IRR has been cleaved and joined to a target just outside the 5′ end of IRL; the remaining vector sequences have a free 5′ end, derived from cleavage at the 3′ end of IRR, and a free 3′ end, released upon cleavage of the target site adjacent to IRL. We propose that figure‐eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co‐integrates.