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N D Grindley

Publications and source records attributed to N D Grindley.

At least 73 records · Page 4Linked to original sources
PubMed1984

Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically.

The 20,500-dalton gamma delta resolvase monomer can be cleaved by chymotrypsin into a 5000-dalton COOH-terminal fragment and a 15,500-dalton NH2-terminal fragment that have been purified. Two crystal forms of the large fragment have been obtained, one of which is isomorphous with crystals of the native protein, showing that the large fragment makes the protein-protein contacts in the crystal and that the small fragment is segmentally disordered relative to the large fragment. Nuclease protection demonstrates that the small fragment binds specifically to all three DNA binding sites protected by resolvase. However, unlike native resolvase, which binds to all three complete sites with equal affinity, the small fragment binds to each of the six half sites with a different affinity. It has not been possible to demonstrate specific DNA binding of the larger fragment. Thus, resolvase has a modular construction analogous to that found for some repressors and activators; its COOH-terminal domain recognizes specific sequences in the DNA and its NH2-terminal domain mediates protein-protein interactions and probably has the enzymatic activity.

Amino Acid Sequence↗
PubMed1984

Replicative and conservative transpositional recombination of insertion sequences.

We have presented the results of experiments with IS903- and IS10- derived transposons that have led us to the following conclusions: The predominant mechanism of transpositional recombination of these IS elements is a donor-suicide process that results intermolecularly in a simple IS insertion. This process presumably involves little or no replication of the IS. Intramolecular transposition by this process normally results in nonviable products. However, in the particular situation where the transpositional target lies within the transposon, viable products are obtained; these are deletions and deletion-inversions. Deletions between an IS and a target lying outside the element (the conventional "adjacent deletion") occur by a fully replicative process analogous to the formation of cointegrate molecules in intermolecular transposition. The ability of an IS to promote adjacent deletions correlates closely with its ability to fuse replicons into a cointegrate. Before transposition can occur, a complex of the transposase and both IS ends is probably formed. Requirement for such a pretranspositional complex is suggested by the effect on transpositional frequency of changing the distance between the ends. Our results do not support any of the asymmetrical models for transposition. They are, however, compatible with a modified version of the symmetric model proposed by Shapiro (1979). It is interesting to note the similarity between the structures generated by intramolecular simple transposition of an inverse transposon and the circular structures apparently formed by retroviral and copia autointegrative transposition. Shoemaker et al. (1981a,b) and Flavell and Ish-Horowicz (1983) have characterized circular molecules from retrovirally infected cells and Drosophila tissue-culture cells, respectively. The structures of some of the circular molecules resemble deletions and deletion-inversions (Fig. 3B). To our knowledge, a circular species containing two long terminal repeats (LTRs) and an adjacent deletion, which we predict could only occur by a fully replicative process given the similarity in geometry of an LTR to an IS, have not been found. It would appear, then, that the molecule containing two LTRs acts as an inverse transposon, integrating into itself. Shoemaker et al. (1981b) and Flavell and Ish-Horowicz (1983) have also suggested that these products arise from molecules containing two LTRs. We suggest that the two inside LTR ends interact in a conservative, intramolecular, simple transpositionlike event.

DNA Replication↗
PubMed1984

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene.

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.

Bacteriophage lambda↗
PubMed1983

Insertion sequence duplication in transpositional recombination.

Insertion sequences (IS) are discrete segments of DNA that can transpose from one genomic site to another and promote genetic rearrangements. A question that is central to understanding the mechanism of transpositional recombination is whether genetic rearrangements are accompanied by duplication of the IS that promotes them. Analysis of adjacent deletions mediated by IS903 provides the strongest evidence to date than any IS-mediated transpositional recombination can occur by an efficient replicative mechanism.

Base Sequence↗
PubMed1983

Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.

Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems.

Cloning, Molecular↗
PubMed1982

Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I.

We report the nucleotide sequence of 3.2 kilobase pair region of the Escherichia coli polA gene, comprising the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S. (1982) J. Biol. Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our sequence. Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem. Biophys. Res. Commun. 37, 982-989; Klenow, H., and Henningsen, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polA1 and wild type alleles, we have identified the polA1 mutation as a change from Trp (TGG) to amber (TAG) at residue 342.

Amino Acid Sequence↗
PubMed1982

Transposon-mediated site-specific recombination: identification of three binding sites for resolvase at the res sites of gamma delta and Tn3.

The tnpR gene product, resolvase, of the transposable element gamma delta mediates site-specific recombination between two copies of gamma delta 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 gamma delta and the related transposon, Tn3, 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.

Binding Sites↗
PubMed1982

Identification of two genes immediately downstream from the polA gene of Escherichia coli.

We have identified two genes within a 1-kilobase region immediately following the polA gene of Escherichia coli. The first, whose transcription is initiated about 150 base pairs beyond the end of the polA coding sequence, is the gene corresponding to the previously sequenced "spot 42 RNA" (B. G. Sahagan and J. E. Dahlberg, J. Mol. Biol. 131:573--592, 1979). The second, located further downstream and transcribed towards polA, is the structural gene for a 22-kilodalton polypeptide, which we have detected by using plasmid-directed protein synthesis in maxicells. Sequence analysis of this region of the E. coli genome suggests that it contains little, if any, redundant DNA.

Bacterial Proteins↗
PubMed1981

Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site.

Resolvase, the product of the tnpR gene of the transposable element gamma delta, mediates a site-specific recombination between two copies of the element directly repeated on the same replicon. The resolution site, res, at which resolvase acts lies in the intercistronic region between the tnpA and tnpR genes. We have studied this site-specific recombination in vitro. In the absence of Mg2+, a resolvase-res complex is formed, which contains DNA molecules that have been cleaved at res. Our data suggest that in this complex resolvase is covalently attached to the 5' ends of the cleaved DNA, leaving free 3' hydroxyl groups. DNA cleavage is stimulated by the interaction of two res sites on the same substrate molecule and appears to be an intermediate step in normal res site recombination. We show that the DNA is cut within a region previously identified as containing the crossover point at the palindromic sequence 5'- (see formula in text) to generate 3' extensions of two bases.

Bacterial Proteins↗
PubMed1980

Genetic and DNA sequence analysis of the kanamycin resistance transposon Tn903.

The kanamycin resistance transposon Tn903 consists of a unique region of about 1000 base pairs bounded by a pair of 1050-base-pair inverted repeat sequences. Each repeat contains two Pvu II endonuclease cleavage sites separated by 520 base pairs. We have constructed derivatives of Tn903 in which this 520-base-pair fragment is deleted from one or both repeats. Those derivatives that lack both 520-base-pair fragments cannot transpose, whereas those that lack just one remain transposition proficient. One such transposable derivative, Tn903 delta I, has been selected for further study. We have determined the sequence of the intact inverted repeat. The 18 base pairs at each end are identical and inverted relative to one another, a structure characteristic of insertion sequences. Additional experiments indicate that a single inverted repeat from Tn903 can, in fact, transpose; we propose that this element be called IS903. To correlate the DNA sequence with genetic activities, we have created mutations by inserting a 10-base-pair DNA fragment at several sites within the intact repeat of Tn903 delta 1, and we have examined the effect of such insertions on transposability. The results suggest that IS903 encodes a 307-amino-acid polypeptide (a "transposase") that is absolutely required for transposition of IS903 or Tn903.

Base Sequence↗
PubMed1979

Transposition of the Escherichia coli insertion element gamma generates a five-base-pair repeat.

We have determined DNA sequences surrounding the termini of the Escherichia coli insertion element gamma delta, both at its normal locus on the F (fertility) factor and at three different sites of insertion into the plasmid pBR322. After transposition, a five-base-pair pBR322 sequence is duplicated and appears in direct orientation adjacent to each end of the element. No such duplication flanks the ends of gamma delta in F, and there is no apparent homology between the sequences surrounding gamma delta in F and the five-base-pair duplications generated by insertion. These findings suggest that the duplications are not essential for transposition and that they do not act to direct gamma delta to a homologous site in the target chromosome. In addition, we find that the 35-base-pair inverted repeat that comprises the termini of gamma delta is strikingly similar in sequence to the ends of both the ampicillin-resistance transposon Tn3 and a 200-nucleotide-long sequence on the plasmid pSC101 which has been shown to mediate recombination with phage f1 replicative form. Within the terminal region, there is a specific heptanucleotide sequence common to each of the above elements and to bacteriophage Mu, all of which generate five-base-pair repeats upon insertion.

Base Sequence↗
PubMed1978

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.

Base Sequence↗
PubMed1976

Mapping of the polA locus of Escherichia coli K12: orientation in the amino- and carboxy-termini of the cistron.

Three mutations of the polA cistron, the structural gene for DNA polymerase I of E. coli, have been ordered by three factor transductional crosses. The three mutant polymerase species have altered properties which may be ascribed to defects located in different portions of the polypeptide chain. Our data indicate that the amino terminal end is encoded by the end of the polA cistron nearer to metE and that transcription and translation proceed clockwise on the E. coli circular map towards the rha locus.

Chromosome Mapping↗
PubMed1976

Effects of different alleles of the E. coli K12 pol A gene on the replication of non-transferring plasmids.

The effects of eight different polA alleles on the replication of six different non-transferring enterobacterial plasmids have been tested. Using phage P1CM transduction, different allelic polA mutations were introduced into E. coli K12 strains carrying one of several antibiotic resistance plasmids. Plasmid stability in the transductants was examined by testing clones for drug resistance after growth under various conditions. From the results, the R factors may be divided into three different classes. One plasmid is only affected by PolA conditions which inhibit host cell growth, 3 plasmids (from the same compatibility group) are unstable under conditions in which the cells are severely deficient in DNA polymerase I and two other plasmids (compatible with each other and with the other 4) are immediately lost from such transductants and are unstable in a number of others. Furthermore, the plasmids which are most dependent on DNA polymerase I have been shown to replicate in the presence of chloramphenicol and therefore typigy a class of plasmids which includes bacteriocinogenic factors such as ColE1 and CloDF13, resistance determinant RSF1030 and the E. coli 15 minicircular plasmid.

Alleles↗
PubMed1976

polA6, A mutation affecting the DNA binding capacity of DNA polymerase I.

The polA6 mutation is an allele of the polA gene of Escherichia coli which produces a DNA polymerase I species readily distinguishable from that produced by the wild type allele. Experiments described here show that this enzyme has an altered pH optimum for polymerization and a lower binding affinity for DNA. The defect clearly lies within the carboxyl-terminal large fragment of the enzyme produced by in vivo or in vitro proteolysis since the fragment has the same pH optimum for polymerization as the intact enzyme. The polA6 enzyme and its fragment are more sensitive to phosphate ions than the wild type polymerase, and the large fragment is less efficient at binding poly d(AT) in in vitro binding assays. Although the specific nucleolytic activity of the polA6 enzyme is higher than that of the wild type, there is no apparent alteration in pH optimum for the hydrolysis of eigher double or single stranded DNA.

Alleles↗