Biomedical subjects

T Kodadek

Publications and source records attributed to T Kodadek.

At least 55 records · Page 3Linked to original sources

PubMed1994

Inhibition of the DNA unwinding and ATP hydrolysis activities of the bacteriophage T4 DDA helicase by a sequence specific DNA-protein complex.

The structural nature of helicase-substrate complexes in the unwinding mode is difficult to study due to their transient nature. We report here a simple method to freeze a DNA helicase at a specific position. The method employs a sequence-specific DNA protein complex as a "roadblock" to helicase movement. The feasibility of the approach is demonstrated by trapping the dda protein of bacteriophage T4 upstream of a GAL4-DNA complex. The presence of the trapped helicase is demonstrated directly by protection of a nearby restriction site and indirectly by the inability of the helicase to recycle rapidly to unwind an unmodified substrate. The half-life of this frozen complex is approximately two minutes under the conditions employed. These results suggest that further study of this novel complex will prove fruitful in elucidating the properties of a DNA helicase in its unwinding mode. As a case in point, it is shown that the dda protein ceases to hydrolyze ATP while stalled, suggesting that nucleotide triphosphate hydrolysis is coupled to translocation for this enzyme.

Adenosine Triphosphatases↗

PubMed1994

Zinc-free and reduced T4 gene 32 protein binds single-stranded DNA weakly and fails to stimulate UvsX-catalyzed homologous pairing.

The functional role of Zn(II) binding by T4 gene 32 protein (gp32), a single-stranded DNA-binding protein, has been investigated by assessing the capacity of a well-characterized metal-free gp32 derivative to function in vitro as an accessory protein of T4 uvsX-catalyzed homologous pairing. Metal-free gp32 was prepared upon reaction of cysteine thiolates with methylmethanethiol-sulfonate to form the mixed disulfide Cys-SSCH3 or S-methylated species. Far and near ultraviolet circular dichroism spectroscopy suggest a moderate but easily detected change in the far UV region, accompanied by only a minor alteration in the near UV region, relative to the Zn(II)-containing protein. Restoration of the wild-type spectral features is accomplished upon the addition of 2 mM dithiothreitol and excess Zn(II) but not dithiothreitol alone. Unlike wild-type gp32, apo S-methylated gp32 shows weak binding to the recombination substrate, single-stranded M13mp19, and fails to stimulate homologous pairing with a linear M13mp19 duplex substrate by uvsX protein. Complete reactivation of the apo S-methylated protein as a recombination-accessory protein is achievable in situ in the presence of reducing agent and sufficient exogenous Zn(II), but not one or the other alone. Analogous results are obtained with S-methylated C166S (Cys166-->Ser) gp32, revealing that only the metal-liganding cysteines participate in the reconstitution. These findings suggest that formation of the Zn(II) chelate is directly linked to single-stranded DNA binding and functional efficacy of gp32 in DNA metabolism.

Apoproteins↗

PubMed1993

How do "Zn2 cys6" proteins distinguish between similar upstream activation sites? Comparison of the DNA-binding specificity of the GAL4 protein in vitro and in vivo.

The GAL4 protein of Saccharomyces cerevisiae is the prototype of a family of transcription factors that contain a "Zn2Cys6" coordination complex in the DNA-binding domain. GAL4 activates the transcription of genes involved in galactose and melibiose metabolism by binding to sites that contain one or more copies of a sequence 5'-CGGN5TN5CCG-3'. Other Zn2Cys6 proteins in S. cerevisiae also recognize sequences containing two CGG triplets, but with different spacings between them. In this report we investigate the mechanism by which GAL4 distinguishes its bona fide binding site from similar sequences as well as from bulk genomic DNA. In vitro, GAL4 recognizes with moderate to high affinity a variety of sites of the general formula (A/C)GGN10-12CCG. This level of specificity is apparently insufficient for the activator to carry out its biological role. However, many of the sites to which GAL4 binds in vitro do not support GAL4-activated transcription in vivo. In most cases there is not a quantitative correlation between the relative affinity of a site for GAL4 in vitro and the level of GAL4-dependent transcription supported by it in vivo. These data imply that there is some mechanism in vivo by which the intrinsic binding specificity of GAL4 is modified.

Base Composition↗

PubMed1993

The role of protein-protein interactions in the assembly of the presynaptic filament for T4 homologous recombination.

The presynaptic filament is an obligatory intermediate in general genetic recombination. It is composed of a strand transferase protein polymerized along single-stranded DNA. In bacteriophage T4-infected cells, the presynaptic filament is composed of at least three proteins. In addition to the strand transferase (the uvsX protein), the uvsY (an accessory factor), and gene 32 (a helix-destabilizing factor), proteins also bind to the single-stranded DNA. In this report, we probe the assembly of the T4 presynaptic filament and the organization of the proteins in the complex. We find that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32 protein-covered DNA. Efficient binding of the uvsY protein to DNA is shown to be necessary for subsequent loading of the uvsX protein.

Adenosine Triphosphatases↗

PubMed1993

The acidic activation domains of the GCN4 and GAL4 proteins are not alpha helical but form beta sheets.

The most common class of activation domains, the so-called acidic activators, has been proposed either to adopt an amphipathic alpha-helical structure or to exist as unstructured "acid blobs." However, genetic analysis of an acidic activation domain in the yeast GAL4 protein has suggested that the structure of the activation region is a beta sheet. To distinguish between these models, we conducted a biophysical analysis of peptides corresponding to the yeast GAL4 and GCN4 acidic activation domains. Circular dichroism spectroscopy shows that the peptides are not alpha helical, but that they can undergo a transition to a structure that is almost 100% beta sheet in character in slightly acidic solution. We also show that the artificial acidic activator AH has structural properties that are markedly different from the natural GAL4 and GCN4 domains and does not adopt a beta-rich structure at reduced pH.

Amino Acid Sequence↗

PubMed1993

How does the GAL4 transcription factor recognize the appropriate DNA binding sites in vivo?

The GAL4 protein of yeast activates the transcription of several genes involved in galactose metabolism. This event requires that GAL4 bind to upstream activation sites with the consensus sequence 5'-CGGN5(T/A)N5CCG-3'. We review the general requirements that must be met for a protein such as GAL4 to find and remain bound to its target site in vivo. Evidence is presented that the GAL4 DNA-binding domain itself has insufficient intrinsic sequence selectivity to fulfill the task of targeting GAL4 to the appropriate sites in a yeast genome. Possible mechanisms by which this selectivity is enhanced in vivo are discussed.

Base Sequence↗

PubMed1992

The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA.

The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed.

Adenosine Triphosphatases↗

PubMed1991

Inhibition of protein-mediated homologous pairing by a DNA helicase.

Protein-mediated exchange of homologous DNA strands is a central reaction in general genetic recombination and the mechanism by which proteins mediate this process in vivo is a topic of keen interest. The dda protein of the bacteriophage T4 is a DNA helicase that has been shown to accelerate branch migration catalyzed by the phage uvsX and gene 32 proteins in vitro (Kodadek, T., and Alberts, B.M. (1987) Nature 326, 312-314). This study did not address the potential role of the helicase in protein-mediated homologous pairing, the first phase of the overall strand-exchange reaction. It is shown here that the dda protein inhibits uvsX protein-mediated pairing between homologous single and double-stranded DNAs. Experiments using deproteinized heteroduplex joints demonstrate that the dda helicase is capable of unwinding these structures to some extent and suggests that this activity may be responsible for the observed inhibition of pairing. It is found that the helicase also reduces the level of uvsX protein-mediated, single-stranded DNA-dependent ATP hydrolysis in the strand-exchange reactions, suggesting that the helicase may also act to destabilize the uvsX protein-DNA filaments that are important intermediates in the pairing reaction. Three other helicases are found to have no effect on the uvsX protein-mediated pairing reaction. A model rationalizing the ability of the dda protein to both inhibit homologous pairing and stimulate branch migration is presented and possible in vivo roles for this interesting activity are discussed.

Adenosine Triphosphate↗

PubMed1991

Spectroscopic studies of the DNA binding site of the GAL4 "zinc finger" protein.

The yeast GAL4 protein, a transcriptional activator of genes involved in galactose metabolism, binds as a dimer to several closely related seventeen base pair upstream activation sequences (UASGs) that are nearly symmetric about a central dT-dA base pair. A previous study of a GAL4-UASG complex (Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F. and Ptashne, M. (1989) J. Mol. Biol. 209, 423-432) elucidated a pattern of contacts consistent with the protein partially wrapping itself around the helical cylinder, assuming a B-form conformation for the DNA. Alternatively, both monomers could sit on one face of the cylinder if the DNA exists in an underwound conformation such as A-form. Spectroscopic studies that distinguish between these models are reported here. Oligonucleotides containing the consensus UASG or a nine base pair "half site" both exhibit circular dichroism (CD) spectra characteristic of B-form DNA. Two-dimensional NMR studies of the half-site also indicate a B-form conformation. When a GAL4 protein fragment containing the entire DNA-binding and dimerization domains (amino acids 1-140) is bound to the UASG, the CD spectrum above 240 nm changes only slightly, and not in a manner consistent with DNA unwinding. Our studies suggest that the UASG does not adopt an unusual underwound conformation in the absence or presence of the GAL4 protein, and favor the model in which the dimer partially wraps around the helix cylinder.

Base Sequence↗

PubMed1990

The role of the bacteriophage T4 gene 32 protein in homologous pairing.

The gene 32 protein of the bacteriophage T4 is required for efficient genetic recombination in infected Eschericia coli cells and strongly stimulates in vitro pairing catalyzed by the phage uvsX protein, a RecA-like strand transferase. This helix-destabilizing factor is known to bind tightly and cooperatively to single-stranded DNA and to interact specifically with the uvsX protein as well as other phage gene products. However, its detailed role in homologous pairing is not well understood. I show here that when the efficiency of uvsX protein-mediated pairing is examined at different gene 32 protein and duplex DNA concentrations, a correlation between the two is found, suggesting that the two interact in a functionally important manner during the reaction. These and other data are consistent with a model in which the gene 32 protein binds to the strand displaced from the recipient duplex during pairing, thereby stabilizing the heteroduplex product. An alternative model in which the gene 32 protein replaces UvsX on the invading strand, thereby freeing the strand transferase to bind to the displaced strand, is also considered.

DNA, Viral↗

PubMed1990

Functional interactions between phage T4 and E. coli DNA-binding proteins during the presynapsis phase of homologous recombination.

The "protein machine" for phage T4 homologous recombination has begun to be assembled in vitro. A particularly heavily studied reaction has been the uvsX protein (a RecA-like strand transferase)-mediated homologous pairing reaction between single and double-stranded DNAs, a key step in the recombination cycle in vivo. A necessary prerequisite for uvsX protein-mediated pairing is the polymerization of this factor along the invading single strand, a process known as presynapsis. Recent work has indicated that at least two other T4 recombination factors are involved in this process as well, the uvsY and gene 32 products. These proteins are also ssDNA-binding factors and exhibit an affinity for UvsX and each other. In order to begin to sort out the potential functional roles played by these protein-protein interactions in presynapsis, I have examined the ability of the uvsX protein to form stable filaments along ssDNA in the presence of these proteins. It is shown that the uvsY protein relieves the inhibition to filament formation due to the presence of the gene 32 protein, but experiments with the E. coli SSB protein (the bacterial analogue of gp32) suggest that this effect does not involve a direct interaction between UvsY and gp32.

DNA-Binding Proteins↗

PubMed1990

Homologous pairing in vitro initiated by DNA synthesis.

A number of models have been proposed for the initiation of general genetic recombination. One of these, originally proposed by Meselson and Radding, imagines that the single-stranded 5' tail that results from strand displacement DNA repair synthesis can initiate homologous recombination by invading a homologous duplex. The resultant D-loop intermediate is then processed into mature products. We demonstrate here that an in vitro system composed of the bacteriophage T4 uvsX protein (a RecA-like "strand transferase") and part of the T4 DNA polymerase holoenzyme efficiently mediates pairing between nicked double-stranded circular and linear duplex DNAs, thereby demonstrating the feasibility of a key step in the Meselson-Radding model.

DNA↗

PubMed1989

The phage T4 uvs Y recombination protein stabilizes presynaptic filaments.

The bacteriophage T4 uvsY protein is required for efficient recombination in T4-infected Escherichia coli cells. Previous in vitro work has shown that the purified uvsY protein is an accessory protein; it stimulates homologous pairing catalyzed by the phage uvsX protein (a RecA-like recombinase) under certain conditions. We show here that this effect can be traced, at least in part, to a UvsY-dependent stabilization of uvsX protein-single-stranded DNA complexes. These presynaptic filaments are one of the early obligatory intermediates in the strand exchange reaction between homologous single- and double-stranded DNAs. The mechanism of filament stabilization seems to involve a slower loss of UvsX subunits. A model that accounts for the data is presented in which both recombination proteins are incorporated into the presynaptic filament.

Adenosine Triphosphatases↗

PubMed1988

The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins.

A strand exchange reaction between a single-stranded DNA circle and a homologous linear double-stranded DNA molecule is catalyzed by a mixture of two T4 bacteriophage proteins, the uvsX protein (a DNA-dependent ATPase that resembles the recA protein) and the gene 32 protein (a helix-destabilizing protein). The products are different from those formed in the corresponding recA protein-catalyzed reaction; rather than producing a linear single strand plus a nicked circular double-stranded (form II) DNA molecule as the final products, interlinked DNA networks are rapidly generated. Electron microscopy reveals that these networks form from multiple pairing reactions that involve the recombination intermediates. Since the uvsX protein is present in substoichiometric quantities, it presumably recycles to catalyze these successive pairing events. Recycling of the uvsX protein has been more directly examined in an assay that monitors the rate of uvsX protein-catalyzed branch migration. The branch migration reaction is rapidly inhibited by dilution of the uvsX protein or by the addition of a heterologous competitor DNA, showing that the uvsX protein-DNA filaments that catalyze strand exchange are dynamic structures. The evidence suggests that individual uvsX protein monomers are continuously entering and leaving the cooperatively formed filament in a cycle that is strongly affected by their ATP hydrolysis.

Adenosine Triphosphatases↗

PubMed1988

Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication.

We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

Base Sequence↗

PubMed1984

Epoxidation of olefins by cytochrome P-450 model compounds: mechanism of oxygen atom transfer.

The mechanism of the Mn(III) porphyrin-catalyzed epoxidation of olefins by lithium hypochlorite is examined. The active oxidant is thought to be a high-valent manganese-oxo complex. It is shown that a relatively stable intermediate is reversibly formed upon interaction of the olefin and the oxo complex. The decomposition of this intermediate to Mn(III) porphyrin and epoxide is the rate-determining step of the catalytic cycle. Some analogies to the biochemical epoxidation of olefins catalyzed by cytochrome P-450 are discussed.

Alkenes↗

PubMed1983

Oxygenation of hydrocarbons by cytochrome P-450 model compounds: modification of reactivity by axial ligands.

The rate of olefin oxygenation catalyzed by synthetic metalloporphyrins is examined, employing sodium hypochlorite as the oxygen atom source. The rate of epoxidation and the stability of the catalyst are shown to be dependent on the nature of the axial ligand employed. A rationale for this effect is presented and analogy is made to the role of the thiolate ligand in cytochrome P-450.

Cytochrome P-450 Enzyme System↗

PubMedDate not supplied

Cleavage of DNA by electrochemically activated MnIII and FeIII complexes of meso-tetrakis (N-methyl-4-pyridiniumyl)porphine.

Electrochemical methods were used to activate MnIII and FeIII complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2TMPyP) to cause cleavage of pBR322 DNA and to study their interaction with sonicated calf thymus DNA. Electrochemical reduction of MnIIITMPyP and FeIIITMPyP (at low concentrations) in the presence of O2 was required to activate these complexes. However, FeIIITMPyP at 1 x 10(-6) M produced DNA strand breakage without being electrochemically reduced. At low concentrations, FeIITMPyP was more efficient at cleaving DNA than MnIITMPyP. Reduction of O2 at a platinum electrode also produced some cleavage but to a much smaller extent. The oxidized form of MnIIITMPyP (charge 5+) has higher affinity for sonicated calf thymus (CT) DNA than the reduced form (charge 4+), as determined by the negative shift in E degrees' for the voltammetric wave in the presence of DNA. Both forms of FeIIITMPyP (charge 4+) interact with DNA to about the same extent. Differential pulse voltammetry was used to determine binding constants (K) and binding-site sizes (s) of the interaction of these metalloporphyrins with sonicated CT DNA. The data were analyzed assuming both mobile and static equilibria. MnIIITMPyP binds to DNA (5 mM Tris, 50 mM NaCl, pH 7) with K = 5 (+/- 2) x 10(6) M-1, s = 3 bp (mobile) or K = 3.6 (+/- 0.3) x 10(6) M-1, s = 4 bp (static). FeIIITMPyP at that ionic strength caused DNA precipitation. At higher ionic strength (0.1 M Tris, 0.1 M NaCl, pH 7), FeIIITMPyP associates to DNA with K = 4.4 (+/- 0.2) x 10(4) M-1, s = 5 bp (mobile) or K = 1.9 (+/- 0.1) x 10(4) M-1, s = 6 bp (static).

DNA↗