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N D Grindley

Publications and source records attributed to N D Grindley.

At least 55 records · Page 3Linked to original sources
PubMed1990

Integration host factor increases the transpositional immunity conferred by gamma delta ends.

The ends of the bacterial transposon gamma delta contain adjacent binding sites for gamma delta transposase and integration host factor (IHF). IHF+ and IHF- strains were used in conjunction with gamma delta transposon ends containing or lacking the site for IHF binding to determine the role that IHF plays in various gamma delta-mediated transposition events. IHF was not essential for the transposition of gamma delta and seemed to decrease its frequency of transposition about threefold. IHF played no role in determining the distribution of gamma delta inserts into a target replicon, nor did it significantly alter the frequency of simple transpositions. The only clear role discerned for IHF and the terminal IHF-binding sites was in transposition immunity. IHF stimulated the immunity of those plasmids that contain an end of gamma delta, provided the end included the terminal IHF-binding site. For both ends, the degree of stimulation of immunity was similar to the stimulation of binding of transposase by IHF.

Bacterial Proteins↗
PubMed1990

Uncoupling of transpositional immunity from gamma delta transposition by a mutation at the end of gamma delta.

The transposon gamma delta, in common with other members of the Tn3 family, confers transpositional immunity, a phenomenon by which plasmids containing a single transposon end show reduced activity as targets for further insertion by the same element. We found that a copy of a mutant delta end, in which the two terminal base pairs (5' GG) were substituted with cytosines, conferred the same degree of immunity as the unaltered delta end. However, a transposon analog with the mutant delta end as its termini could not transpose. These results suggest that the binding of transposase to a site on a target replicon is sufficient to confer immunity and that immunity does not involve subsequent DNA transactions at the bound target site, analogous to the catalytic processes that occur at the transposon ends during transposition.

Base Composition↗
PubMed1989

Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase.

The ability to determine protein structures by X-ray crystallography is often thwarted by the difficulty of finding isomorphous heavy-atom derivatives. The crystal structure of the site-specific recombinase, resolvase, has been difficult to determine for this reason. We have overcome this problem by introducing 13 single cysteine substitutions into the resolvase catalytic domain using oligonucleotide mutagenesis. The mutant proteins were screened for their ability to crystallize into the orthorhombic form and bind mercury ions isomorphously. Two mutant proteins provided excellent heavy-atom derivatives. This approach should be of general use and particularly helpful in cases where traditional methods have failed to produce a derivative.

Amino Acid Sequence↗
PubMed1989

The 43 residue DNA binding domain of gamma delta resolvase binds adjacent major and minor grooves of DNA.

The carboxyl-terminal domain of gamma delta resolvase binds to each half of the three resolvase binding sites that constitute the recombination site, res. Ethylation inhibition experiments show that the phosphate contacts made by the C-terminal DNA binding domain are similar to those made by intact resolvase, with the exception of a single phosphate at the inside end of each contact region which is contacted solely by the intact resolvase. The DNA binding domain makes essentially identical contacts to all 6 half sites, whereas the intact resolvase makes slightly different contacts to each binding site. Despite its small size, only 43 amino acid residues, the resolvase C-terminal domain interacts with an unusually large segment of DNA. Phosphate contacts extend across an adjacent major and minor groove of DNA and about one third of the circumference around the helix. The minimal binding segment, determined experimentally, is a 12 bp sequence that includes the 9 base pair inverted repeat (common to all half sites), the adjacent 3 base pairs (towards the center of the intact resolvase binding site), and phosphates at both ends.

Amino Acid Sequence↗
PubMed1988

Uncoupling of the recombination and topoisomerase activities of the gamma delta resolvase by a mutation at the crossover point.

In several well-characterized site-specific recombination systems it has been shown that, for efficient recombination, the two recombining sites must have identical DNA sequences across the region between the staggered points of exchange. The precise DNA sequence of this overlap region, however, appears to be of little importance (with the exception of one position in the loxP site of bacteriophage P1 (ref. 6]. In this report we characterize a mutant recombination site for the site-specific recombination enzyme gamma delta resolvase (encoded by the gamma delta transposon), in which the dinucleotide at the crossover point is changed from AT to CT. Our results indicate that identity of the two overlap regions is not sufficient for recombination. Although resolvase binds normally to the mutant site and induces the structural deformation characteristic of the wild-type recombination site, catalysis at the crossover point (cutting and rejoining of DNA strands) is effectively limited to just one of the two strands, allowing resolvase to act as a topoisomerase but not as a recombinational enzyme.

Base Sequence↗
PubMed1988

Gamma delta transposase and integration host factor bind cooperatively at both ends of gamma delta.

gamma delta, a prokaryotic transposon, encodes a transposase that is essential for its transposition. We show here, by DNase I protection experiments, that purified gamma delta transposase binds at the transposon's inverted repeats (IRs). Immediately adjacent to each transposase binding site (and within gamma delta DNA) we have identified a binding site for an additional protein factor, the Escherichia coli-encoded integration host factor (IHF). The binding of transposase and IHF to these adjacent sites is mutually cooperative. An IHF binding-site was also found in the original target DNA, just outside one of the ends of gamma delta. The affinity of IHF for this flanking site is reduced by transposase. These results demonstrate that gamma delta transposase binds at the IRs of gamma delta, and suggest that IHF may be involved in forming a transposase-DNA complex and/or influencing the target site selection during the transposition of gamma delta.

Adenosine Triphosphate↗
PubMed1988

The gamma delta resolvase bends the res site into a recombinogenic complex.

We have characterized complexes between the gamma delta resolvase and its recombination site, res, using both a gel retardation assay and DNase I cleavage. The mobility of resolvase-res complexes in polyacrylamide gels is sensitive to the location of res within the DNA fragment and is at a minimum when res is at its center. This behavior is characteristic of a protein-dependent bend. By the same assay we have found that bends are induced upon the binding of resolvase to each of the three individual binding sites that constitute res. In the wild-type res, the centers of binding sites I and II are 53 bp apart and the central section of the intersite DNA is sensitive to DNase I cleavage. We find that insertions of 10 or 21 bp (one or two turns of the DNA helix) have no discernible effect on the ability of res to recombine or to form complexes with resolvase. However, insertions of short segment (e.g. 6 or 17 bp) equivalent to nonintegral numbers of helical turns, inhibit recombination and prevent the formation of the normally compact resolvase-res complex. Complexes of resolvase with res containing 10 or 21 bp insertions exhibit a pattern of enhanced and suppressed DNase I cleavages that suggest that the intersite segment is curved. This curvature requires both that site I and II are appropriately spaced, and that site III is also present and occupied.

Binding Sites↗
PubMed1987

Helical phasing between DNA bends and the determination of bend direction.

The presence and location of bends in DNA can be inferred from the anomalous mobility of DNA fragments or protein-DNA complexes during electrophoresis in polyacrylamide gels. Direction of bending is not so easily determined. We show here that a protein-induced bend, when linked to a protein-independent DNA bend by a segment of variable length, exhibits an electrophoretic mobility that varies in a sinusoidal manner with the length of the linker. Mobility minima occur once for each addition to the linker of one helical turn of DNA. Since minima should occur when two bends reinforce one another, the direction of one bend relative to the other can be determined from the distances between the two centers of bending at which minima occur. Our results strongly support the idea that the A5-6 tracts in kinetoplast DNA bend towards the minor groove while the bend at the recombination site of the gamma delta resolvase (binding site I of the gamma delta res site) bends towards the major groove.

Animals↗
PubMed1987

The gamma delta 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.

Crossing Over, Genetic↗
PubMed1987

Contacts between gamma delta resolvase and the gamma delta res site.

We have investigated the interaction between resolvase and the res site of the transposon gamma delta by methylation and ethylation interference experiments. We have examined the effect of these DNA modifications both on binding and resolution in vitro. Major groove methylations within a 9 bp sequence that borders each site inhibit binding of resolvase to that site. Ethylation of certain phosphates within, and adjacent to, this border sequence inhibits binding. Together, these interference points define a contact region, present at all three res sites. In vitro resolution is inhibited only by modifications within site I. Inhibition of resolution by methylation of adenines at the center of site I suggests that minor groove contacts near the crossover may be required for resolution activity.

Alkylation↗
PubMed1987

Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats.

The insertion sequence IS903 has perfect, 18-base-pair terminal repeats that are the presumed binding sites of its transposase. We have isolated mutations throughout this inverted repeat and analyzed their effect on transposition. We show that every position in the inverted repeat (with the possible exception of position 4) is important for efficient transposition. Furthermore, various substitutions at a single position can have a wide range of effects. Analysis of these hierarchical effects suggests that transposase contacts the minor groove in the region from position 13 to position 16 but makes major groove (or more complex) interactions with the outer portion of the inverted repeat. Our data indicate that the transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition of virtually all mutant inverted repeats can be rescued by a wild-type end. However, this rescue exhibits a pronounced position effect; in most cases, it is efficient only when the wild-type end is close to the 3' end of the transposase gene. This confirms the cis-acting nature of the transposase protein and suggests the initial transposase-inverted repeat interaction is the rate-limiting step in transposition. From the behavior of transposons with one mutant and one wild-type end, we infer that the inverted repeat contains two functional domains--one for initial complex formation with transposase and the other for effective completion of transpositional recombination. To support this hypothesis we show that an end with a mutation in one domain can significantly rescue an end with a mutation in the other domain.

Binding Sites↗
PubMed1986

A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides.

A method is described for the efficient saturation mutagenesis of a segment of DNA. A pool of mixed oligodeoxynucleotides (oligos) is generated in a single synthesis by deliberately contaminating each nucleotide reservoir with a low concentration of the other three monomers. The concentration at which a particular level of substitution (i.e. single, double, etc.) is favored can be calculated from simple probability. The pool of single-stranded, mixed oligos is then cloned directly into a double-stranded vector. This is achieved by designing the oligo such that it has ends which are complementary to those left by restriction enzymes generating 5' and 3' overhanging ends. The efficiency of the cloning procedure that we describe is greater than 90%. As a result, direct sequence analysis of transformant clones is justified. In the example described, 2/3 of M13 plaques containing an inserted oligo carried one or more mutations. Mutations generated by this method are randomly distributed throughout the inserted DNA and include all types of substitution. The ease and efficiency of the procedure eliminate any requirement for genetic selection of mutants and thus allow phenotypically silent mutations to be obtained.

Cloning, Molecular↗
PubMed1986

Analysis of gamma delta resolvase mutants in vitro: evidence for an interaction between serine-10 of resolvase and site I of res.

The resolvase encoded by the transposon gamma delta mediates a site-specific recombination between the two copies of gamma delta in a cointegrate molecule to yield the final products of transposition. Several mutants of resolvase that lack recombinational activity have been isolated previously, and one of these, which has a serine-to-leucine change at position 10, we have characterized in vitro. We also have constructed a serine-to-cysteine change at position 10 by in vitro mutagenesis and have analyzed this mutant protein in vitro. We find that the cysteine-10 mutant is defective in recombinational activity but binds to the recombinational site, res, similarly to wild-type, as assayed by gel electrophoresis of the protein-DNA complexes. In contrast, the leucine-10 mutant has a specific defect in complex formation with site I, which contains the recombinational crossover point, although it can bind and provide the ancillary functions of resolvase at sites II and III. It has been shown that a phosphoserine linkage is made to the DNA during recombination; because serine-10 is absolutely conserved amongst the family of homologous site-specific recombination proteins, it is a good candidate for the active-site serine. The properties of these resolvase mutants with substitutions at position 10 are consistent with this hypothesis and suggest that serine-10 is in close contact with the DNA at site I.

Amino Acid Sequence↗
PubMed1985

Genetic mapping and DNA sequence analysis of mutations in the polA gene of Escherichia coli.

DNA polymerase I of Escherichia coli provides an excellent model for the study of template-directed enzymatic synthesis of DNA because it is a single subunit enzyme, it can be obtained in large quantities and the three-dimensional structure of the polymerizing domain (the Klenow fragment) has recently been determined (Ollis et al., 1985). One approach to assigning functions to particular portions of the structure is to correlate the altered enzymatic behavior of mutant forms of DNA polymerase I with the change in the primary sequence of the protein. Towards this end we have developed a rapid procedure for mapping any polA mutation to a region no larger than 300 base-pairs within the polA gene. Two series of polA deletion mutants with defined end-points were constructed in vitro and cloned into bacteriophage lambda. These phages can then be used to map precisely E. coli polA mutants. Twelve polA- alleles have been mapped in this way and for nine of them the nature of the mutational change has been determined by DNA sequence analysis. Two of the mutations, polA5 and polA6, which affect the enzyme-DNA interaction, provide evidence for the location of the DNA binding region on the polymerase three-dimensional structure.

Base Sequence↗
PubMed1984

Analysis of the gamma delta res site. Sites required for site-specific recombination and gene expression.

The gamma delta resolvase, product of the transposon's tnpR gene, mediates a site-specific recombination between two copies of gamma delta directly repeated on the same replicon. The site at which the recombination occurs, res, lies between the tnpA and tnpR genes. Within this same region lie the promoters for expression of tnpA and tnpR and the operator sites through which resolvase regulates the transcription. In order to determine the extent of the res site we have constructed in vitro a series of deletions that terminate within the tnpA-tnpR intergenic region, and have analyzed their effect on site-specific recombination. Our results indicate that a fully functional res site is about 115 base-pairs (bp) and runs from a position 15 bp to the left (tnpA-proximal) side of the crossover point to 100 bp to the right. This segment corresponds precisely to the region defined by the three resolvase binding sites that we have demonstrated previously. Alterations of the nucleotide sequence around the crossover point indicate the importance of all or part of the central palindrome 5' T-T-A-T-A-A within which the breakage--reunion reaction takes place. Taken together, our results strengthen our earlier conclusion that resolvase recognizes the 9 bp segment 5' T-G-T-C-Y-N-N-T-A that occurs (in slightly degenerate form) in each half of the three binding sites. Using the deletions we have confirmed that the tnpA promoter spans the crossover site and have shown that the major tnpR promoter in vivo coincides with resolvase binding site II, although a second promoter for tnpR transcription lies across site I.

Base Sequence↗
PubMed1984

Mutants of the gamma delta resolvase: a genetic analysis of the recombination function.

The resolvase protein encoded by the gamma delta transposon has two functions. It catalyzes a site-specific recombination, and it negatively regulates the expression of two transposon genes. Both functions involve the action of resolvase at the res site. To define regions of resolvase that are involved specifically in the recombination reaction, we have isolated and characterized mutants that are defective in cointegrate resolution but retain the ability to bind to res (as measured by regulatory activity). Nine independent mutants were found to contain six different amino acid substitutions among just four distinct residues. The altered residues all lie within the 140 amino acid amino-terminal domain of resolvase and fall within two clusters of amino acids that are highly conserved in other related recombinases. The regulatory properties of the mutants suggest that one of these clusters may be involved in the interaction of the catalytic domain with the crossover site.

DNA Transposable Elements↗