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Biomedical subjects

H A Nash

Publications and source records attributed to H A Nash.

At least 37 records · Page 2Linked to original sources
PubMed1996

A eukaryotic enzyme that can disjoin dead-end covalent complexes between DNA and type I topoisomerases.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

Binding Sites↗
PubMed1996

Influence of general anesthetics on a specific neural pathway in Drosophila melanogaster.

The neural pathway that governs an escape response of Drosophila to sudden changes in light intensity can be artificially induced by electrical stimulation of the brain and monitored by electrical recording from the effector muscles. We have refined previous work in this system to permit reliable ascertainment of two kinds of response: (i) a short-latency response that follows from direct excitation of a giant fiber neuron in the interior of the fly brain and (ii) a long-latency response in which electrical stimulation triggers neurons in the optic ganglia that ultimately impinge on the giant fiber neuron. The general anesthetic halothane is reported here to have very different potencies in inhibiting these two responses. The long-latency response is obliterated at concentrations similar to those that cause gross behavioral effects in adult flies, whereas the short-latency response is only partially inhibited at doses that are 10-fold higher. Three other volatile anesthetic agents show a similar pattern. Thus, as in higher organisms, the Drosophila nervous system is differentiated into components of high and low sensitivity to general anesthetics. Moreover, this work shows that one of the sensitive components of the nervous system lies in the optic lobe and is readily assayed by its effect on downstream systems; it should provide a focus for exploring the effects of genetic alteration of anesthetic sensitivity.

Anesthetics, General↗
PubMed1996

Architectural flexibility in lambda site-specific recombination: three alternate conformations channel the attL site into three distinct pathways.

BACKGROUND: In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo, recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5-10-fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro. RESULTS: We find that IHF can stimulate the in vitro recombination between two attLs that are modified to be defective in one of the high affinity binding sites for Int, P'1. With such substrates, the efficiency of IHF-stimulated recombination is comparable to that seen in vivo. The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo, but that some unknown factor or condition permits it to operate on wild-type as well as mutated attL sites. CONCLUSIONS: The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P'1 site. In addition to demonstrating the architectural flexibility of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.

Attachment Sites, Microbiological↗
PubMed1995

Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target.

We have quantitatively evaluated the affinity of a set of target sites for the integration host factor (IHF) protein of Escherichia coli by their performance as competitors in an electrophoretic mobility shift assay. We also determined how well each of these sites is filled by IHF in vivo. The data show that several natural sites have an affinity not much greater than that required for intracellular occupancy. The data also indicate that very little of the IHF in a cell is present as free protein available for binding, suggesting that binding to non-specific targets dominates the operation of this system. The correlation between in vitro affinity and in vivo occupancy provides a ready means to assess the likely physiological significance of putative IHF sites. It also provides a general method to assess the importance of non-specific interactions by DNA binding proteins inside a cell.

Bacterial Proteins↗
PubMed1995

Suicide substrates reveal properties of the homology-dependent steps during integrative recombination of bacteriophage lambda.

BACKGROUND: A fundamental feature of bacteriophage lambda site-specific recombination is the strict requirement for a region of sequence identity between recombining DNA duplexes. It has been difficult to understand how the recombination machinery identifies and responds to nonhomologies as subtle as a single base-pair substitution, because the reaction intermediates are transient and there are likely to be several different homology-dependent steps. In order to understand better how the recombination machinery compares parental sequences, we have used the recently developed 'suicide substances'--DNA containing 5'-bridging phosphorothioate linkages--to monitor the timing of homology-sensing relative to the strand cleavage reactions. RESULTS: The cleavage reactions for the two different strands of attB, the bacterial recombination locus for lambda integration, show very different degrees of dependence on homology with the partner locus, attP. Strand cleavage at the B binding site for Int recombinase is insensitive to homology. In contrast, cleavage at the B' binding site strongly depends on homology in the three base pairs adjacent to the B site. Strand cleavage at the B site is apparently required for the readout of this homology but, surprisingly, joining of the cleaved B site to a partner is not. CONCLUSIONS: Our finding that cleavage at the B site is insensitive to homology shows that effective synapsis between partners does not depend on sequence matching. Cleavage at the B' site provides the earliest positive signal for a homology-dependent switch in the lambda recombination machinery. Because this switch can occur in the absence of strand joining, the results argue against models that invoke strand ligation as the critical element of homology-sensing. Alternative mechanisms are presented that involve varieties of non-covalent strand swapping. A synthesis of the present results and other recent experiments highlights the importance of the disannealing of complementary strands and their reannealing to new partners, a process traditionally described as branch migration. The reversibility of branch migration and its bias away from mismatched combinations are proposed to be the major mechanisms of homology-sensing during lambda integration.

Bacteriophage lambda↗
PubMed1995

A novel suicide substrate for DNA topoisomerases and site-specific recombinases.

DNA topoisomerases and DNA site-specific recombinases are biologically important enzymes involved in a diverse set of cellular processes. We show that replacement of a phosphodiester linkage by a 5'-bridging phosphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although the bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligation reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lambda integrative recombination. The phosphorothioate substrates offer advantages over conventional suicide substrates, may be potent tools for inhibition of the relevant cellular enzymes and represent a unique tool for the study of many other phosphoryl transfer reactions.

Animals↗
PubMed1995

Mutations that affect ion channels change the sensitivity of Drosophila melanogaster to volatile anesthetics.

We have quantitated the response of D. melanogaster to general anesthetics with a device, the inebriometer, that assays the fly's geotactic and postural behavior. When alleles of several loci that encode or regulate subunits of ion channels were compared with control stocks, several ion channel mutants clearly increased the anesthetic sensitivity of Drosophila. The effects were specific in that: a) for several alleles, genetic tests indicated that the anesthesia phenotype was due to the ion channel mutation and not to extraneous genetic differences between the stocks; b) a given ion channel mutation often affected the response to one anesthetic but not another; and c) the behavior of decapitated flies in the inebriometer indicated that the anesthetic phenotype of several mutants did not merely reflect a global change in the fly's physiology. These results provide support for the idea that ion channels are on the pathway(s) influenced by anesthetics and that different anesthetics use different pathways. They also provide perspective on the behavior of previously isolated mutations (har) that decrease the sensitivity of Drosophila to anesthetics in the inebriometer.

Anesthetics, Inhalation↗
PubMed1994

Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA.

Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact > 25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.

Amino Acid Sequence↗
PubMed1994

Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins.

Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination. HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity. We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase. The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA. We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure. We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome. HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions. These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts. Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA. While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies. This probably reflects subtle structural differences between the assembled complexes.

Allosteric Regulation↗
PubMed1994

Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins.

BACKGROUND: Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions. In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA. IHF is a dimer of non-identical subunits, each about 90 amino acids long. One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site. Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction. RESULTS: We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro. Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers. We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences. CONCLUSIONS: Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically. The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate. We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp). This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface. We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins. In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

Bacterial Proteins↗
PubMed1994

Use of Drosophila mutants to distinguish among volatile general anesthetics.

The way general anesthetics cause their clinically useful effects on the nervous system is not known. Since the principal determinant of the potency of these agents is their solubility in oil, the role of chemical structure in affecting anesthetic targets has been obscured. In this work, we use an intense beam of light (Tinklenberg, J. A., Segal, I. S., Tianzhi, G. & Maze, M. (1991) Ann. N.Y. Acad. Sci. 625, 532-539) to assess the effect of general anesthetics on the capacity of fruit flies to sense a noxious stimulus and respond to it. By examining the effect of halothane-resistant (har) mutations on the potency of various volatile anesthetics in this assay, we establish similarities and differences between these agents that highlight the was chemical structure influences anesthetic action. In general, the potencies of anesthetics with different chemical structure are affected to different extents by one or more har mutations. However, three anesthetics of related structure (enflurane, isoflurane, and desflurane) show quantitatively indistinguishable alterations in potency in each of four genetic tests. These results not only identify important structural features of anesthetics but also place limits on the classical view that all anesthetics act at a common target.

Animals↗
PubMed1993

Characterization of a set of integration host factor mutants deficient for DNA binding.

Integration host factor, IHF, is a sequence-specific DNA-binding and DNA-bending protein composed of two related but non-identical subunits. We report the isolation and characterization of hydroxylamine-induced loss-of-function mutations in the genes encoding the IHF subunits. To screen for mutants that preserve proper folding of IHF, clarified extracts were prepared from each mutant and were assayed for production of each subunit by immunoblotting and for formation of heterodimers by chemical cross-linking and subsequent immunoblotting. Extracts from mutants that met these criteria were found to bind a specific IHF site weakly if at all. These alleles therefore identify candidates for residues that may affect the DNA-binding surfaces of IHF. When projected onto the known tertiary structure of the closely related HU protein, these residues are found at the surface; however, with the exception of a single residue, different regions of the protein are implicated in each subunit. This suggests that, despite their homology, each subunit of IHF directs DNA recognition and binding in a distinct manner. To confirm the significance of the differential location of these mutations, we introduced in each subunit alterations that had been isolated as loss-of-function mutations at the corresponding position in the other subunit. In general, the engineered mutants have phenotypes that are strikingly different from those of their hydroxylamine-induced counterparts. In particular, most of the site-directed mutant IHF proteins form or maintain IHF:DNA complexes more readily than mutants that have the same change in the other subunit and were isolated as loss-of-function mutants. We discuss the positions of the mutant amino acid residues as they relate to a proposed molecular model of an IHF:DNA complex.

Amino Acid Sequence↗
PubMed1993

Synaptic intermediates in bacteriophage lambda site-specific recombination: integrase can align pairs of attachment sites.

Bacteriophage lambda uses site-specific recombination to move its DNA into and out of the Escherichia coli genome. The recombination event is mediated by the recombinase integrase (Int) together with several accessory proteins through short specific DNA sequences known as attachment sites. A gel mobility shift assay has been used to show that, in the absence of accessory proteins, Int can align and hold together two DNA molecules, each with an attachment site, to form stable non-covalent 'bimolecular complexes'. Each attachment site must have both core and arm binding sites for Int to participate in a bimolecular complex. These stable structures can be formed between pairs of attL and attP attachment sites, but cannot include attB or attR sites; they are inhibited by integration host factor (IHF) protein. The bimolecular complexes are shown to represent a synaptic intermediate in the reaction in which Int protein promotes the IHF-independent recombination of two attL sites. These complexes should enable a detailed analysis of synapsis for this pathway.

Bacteriophage lambda↗
PubMed1993

Drosophila melanogaster as a model for study of general anesthesia: the quantitative response to clinical anesthetics and alkanes.

Using an assay of the geotactic behavior of the fruit fly, we have quantified anesthetic effects in Drosophila melanogaster. We determined dose-responses for nine conventional anesthetics and a series of n-alkanes. For the conventional anesthetics, anesthetic potency and olive oil/gas partition coefficients are well correlated. However, as one ascends the series of alkanes of chain lengths 5-11, the increase in anesthetic potency is considerably less than that predicted by olive oil solubility. These examples of agreement and disagreement with the Meyer-Overton rule provide a basis for comparing the responses of fruit flies with those of higher organisms; such comparisons suggest that the anesthetic target is evolutionarily conserved. For alkanes with chain lengths greater than 11, anesthetic effects are variable and slow to develop. This behavior reflects limited volatility and provides no evidence for a cut-off in the action of alkanes at their target.

Alkanes↗
PubMed1992

Deformation of DNA during site-specific recombination of bacteriophage lambda: replacement of IHF protein by HU protein or sequence-directed bends.

Escherichia coli IHF protein is a prominent component of bacteriophage lambda integration and excision that binds specifically to DNA. We find that the homologous protein HU, a nonspecific DNA binding protein, can substitute for IHF during excisive recombination of a plasmid containing the prophage attachment sites attL and attR but not during integrative recombination between attP and attB. We have examined whether IHF and HU function in excisive recombination is mediated through DNA bending. Our strategy has been to construct chimeric attachment sites in which IHF binding sites are replaced by an alternative source of DNA deformation. Previously, we demonstrated that properly phased bends can substitute for the binding of IHF at one site in attP. Although this result is highly suggestive of a critical role of IHF-promoted bending in lambda integration, its interpretation is obscured by the continued need for IHF binding to the remaining IHF sites of these constructs. In the present work, we engineered a population of sequence-directed bends in the vicinity of the two essential IHF sites found in attR and attL. Even in the absence of IHF or HU, pairs of these attachment sites with properly phased bends are active for both in vitro and in vivo excision. This success, although tempered by the limited efficiency of these systems, reinforces our interpretation that IHF functions primarily as an architectural element.

Attachment Sites, Microbiological↗
PubMed1992

Symmetry in the mechanism of bacteriophage lambda integrative recombination.

During the strand-exchange events of bacteriophage lambda integration, pairs of phosphodiester bonds are broken and then rejoined to form novel DNA linkages. The reaction proceeds in vitro in the absence of an external energy source; the bond energy needed to rejoin broken strands of DNA must therefore be conserved during cleavage. Although some of this conservation involves a covalent intermediate between DNA and the recombinase Int, it is possible that such an intermediate is formed with only one of the two phosphodiesters. In such an asymmetric mechanism, the second phosphodiester would be attacked by a nucleophile that is exposed by cleavage of the first DNA strand. In contrast, a symmetric mechanism hypothesizes nucleophilic attack by Int on both phosphodiesters. We have distinguished these two mechanisms by removing potential nucleophiles from the integrative recombination reaction. Our data are inconsistent with an asymmetric mechanism. We conclude that during strand exchange both phosphodiesters proceed through a covalent protein-DNA intermediate.

Bacteriophage lambda↗
PubMed1991

Behavior of a cross-linked attachment site: testing the role of branch migration in site-specific recombination.

Integrative recombination of bacteriophage lambda requires perfect homology between partners over a short segment of DNA, the overlap region, that separates the positions of top and bottom strand exchange. We constructed a specific cross-link between complementary strands in the overlap region of one partner, using a method designed to introduce minimal distortion of DNA. The modified attachment site could initiate recombination, forming a Holliday junction, but could not resolve this junction so as to complete the recombination. This demonstrates that the ability of complementary base-pairs to dissociate is important for overlap region function and strongly supports the view that branch migration across this region is the way homology is sensed during integrative recombination.

Bacterial Proteins↗