Biomedical subjects

T Kodadek

Publications and source records attributed to T Kodadek.

At least 37 records · Page 2Linked to original sources

PubMed1998

Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions.

BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters. The most common type of cooperativity is supported by direct protein-protein interactions. Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro. probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site. This mechanism does not require that the transcription factors have activation domains. We have examined whether this phenomenon occurs in vivo. RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances. In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains. CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates. These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

Bacterial Proteins↗

PubMed1998

Mechanistic parallels between DNA replication, recombination and transcription.

The multiprotein complexes that mediate replication, transcription and homologous recombination in eukaryotic cells face many of the same molecular challenges. These include the recognition of DNA sites embedded in large chromatinized genomes, the denaturation of duplex DNA, and partial dissociation and reassociation at different stages of the catalytic cycle. Therefore, it is not surprising that several steps in the respective catalytic cycles are strikingly similar at the DNA level and may proceed by similar mechanisms. Some of these relationships are reviewed here. It is argued that speculation based on such 'crosspathway' comparisons may be a valuable paradigm for the design of new experiments.

DNA Replication↗

PubMed1998

Protein cross-linking mediated by metalloporphyrins.

A biomimetic protein cross-linking reaction is described which employs oxidatively-activated manganese and iron porphyrins as the reactive species. A wide range of proteins cross-link under these conditions, but only if they are intimately associated in solution. The reaction is rapid, efficient, and will be useful for the suprastructural analysis of multiprotein complexes.

Chelating Agents↗

PubMed1998

Small-molecule-based strategies for controlling gene expression.

A central goal in chemical biology is to gain control over biological pathways using small molecules, and the mRNA-synthesizing machinery is a particular important target. New advances in our understanding of transcriptional regulation suggests strategies to manipulate these pathways using small molecules.

Animals↗

PubMed1997

The gene 32 single-stranded DNA-binding protein is not bound stably to the phage T4 presynaptic filament.

A central reaction in homologous recombination is synapsis, which involves invasion of duplex DNA by a homologous single strand. A key intermediate in this process is the presynaptic filament, a protein-DNA complex composed of a "strand transferase" polymerized along the invading single strand. In this report, the organization and mechanism of assembly of the bacteriophage T4 presynaptic filament are explored. Three T4 proteins, encoded by the uvsX, uvsY and 32 genes, are involved in this process. It is demonstrated that a well-defined series of events involving multiple protein-DNA and protein-protein interactions is required to mediate a transition from an initial gene 32-DNA complex to a mature presynaptic filament in which the UvsX and UvsY proteins are in contact with the DNA and each other, while most or all of the gene 32 protein is removed from the complex.

Bacteriophage T4↗

PubMed1997

DMC1 functions in a Saccharomyces cerevisiae meiotic pathway that is largely independent of the RAD51 pathway.

Meiotic recombination in the yeast Saccharomyces cerevisiae requires two similar recA-like proteins, Dmc1p and Rad51p. A screen for dominant meiotic mutants provided DMC1-G126D, a dominant allele mutated in the conserved ATP-binding site (specifically, the A-loop motif) that confers a null phenotype. A recessive null allele, dmc1-K69E, was isolated as an intragenic suppressor of DMC1-G126D. Dmc1-K69Ep, unlike Dmc1p, does not interact homotypically in a two-hybrid assay, although it does interact with other fusion proteins identified by two-hybrid screen with Dmc1p. Dmc1p, unlike Rad51p, does not interact in the two-hybrid assay with Rad52p or Rad54p. However, Dmc1p does interact with Tid1p, a Rad54p homologue, with Tid4p, a Rad16p homologue, and with other fusion proteins that do not interact with Rad51p, suggesting that Dmc1p and Rad51p function in separate, though possibly overlapping, recombinational repair complexes. Epistasis analysis suggests that DMC1 and RAD51 function in separate pathways responsible for meiotic recombination. Taken together, our results are consistent with a requirement for DMC1 for meiosis-specific entry of DNA double-strand break ends into chromatin. Interestingly, the pattern on CHEF gels of chromosome fragments that result from meiotic DNA double-strand break formation is different in DMC1 mutant strains from that seen in rad50S strains.

Cell Cycle Proteins↗

PubMed1996

Direct association between the yeast Rad51 and Rad54 recombination proteins.

The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH2-terminal 115 residues of the Rad54 protein are necessary for this interaction. Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.

Binding Sites↗

PubMed1996

New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent.

BACKGROUND: To study very large macromolecular complexes, it would be useful to be able to incorporate probe molecules, such as fluorescent tags or photoactivatable crosslinkers, into specific sites on proteins. Current methods for doing this use relatively large amounts of highly purified protein, limiting the general utility of these approaches. The need for covalent posttranslational chemistry also makes it extremely difficult to use modified proteins in studies of native complexes in crude lysates or in living cells. We set out to develop a protein tag that would circumvent these problems. RESULTS: A very simple type of molecular recognition, metal-ligand complexation, can be used to deliver a nickel-based crosslinking reagent to proteins containing a six-histidine (His6) tag. When activated with a peracid, the His6-Ni complex mediates oxidative crosslinking of nearby proteins. The crosslinking reaction does not involve freely diffusible intermediates, and thus only those proteins in close proximity to the His6-tagged polypeptide are crosslinked. CONCLUSIONS: The His6 tag, commonly used as an affinity handle for the purification of recombinant proteins, can also be used as an internal receptor for an oxidative protein-crosslinking reagent. No covalent protein modifications are necessary, since the His6 tag is introduced at the DNA level. The crosslinking reaction is fast, efficient in most cases, and provides products that are easily separated from most other proteins present. This methodology should find widespread use in the study of multiprotein complexes.

Binding Sites↗

PubMed1995

The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo.

Most proteins that activate RNA polymerase II-mediated transcription in eukaryotic cells contain sequence-specific DNA-binding domains and "activation" regions. The latter bind general transcription factors and/or coactivators and are required for high-level transcription. Their function in vivo is unknown. Since several activation domains bind the TATA-binding protein (TBP), TBP-associated factors, or other general factors in vitro, one role of the activation domain may be to facilitate promoter occupancy by supporting cooperative binding of the activator and general transcription factors. Using the GAL4 system of yeast, we have tested this model in vivo. It is demonstrated that the presence of a TATA box (the TBP binding site) facilitates binding of GAL4 protein to low- and moderate-affinity sites and that the activation domain modulates these effects. These results support the cooperative binding model for activation domain function in vivo.

Base Sequence↗

PubMed1995

A single GAL4 dimer can maximally activate transcription under physiological conditions.

Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner. A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill. In this model, various combinations of protein-protein interactions control the level of gene expression. However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo. Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding. These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon.

Base Sequence↗

PubMed1995

Phage T4 homologous strand exchange: a DNA helicase, not the strand transferase, drives polar branch migration.

Homologous strand exchange is a central step in general genetic recombination. A multiprotein complex composed of five purified bacteriophage T4 proteins (the products of the uvsX, uvsY, 32, 41, and 59 genes) that mediates strand exchange under physiologically relevant conditions has been reconstituted. One of these proteins, the product of the uvsY gene, is required for homologous pairing but strongly inhibits branch migration catalyzed by UvsX protein, the phage RecA analog. Branch migration is completely dependent on the gene 41 protein, a DNA helicase that also functions in phage replication. The helicase is delivered to the strand exchange complex by the gene 59 accessory protein in a strand-specific fashion through direct interactions between the gene 59 and gene 32 proteins. These data suggest that strand transferases such as UvsX protein are essential for homologous pairing in vivo, but that a DNA helicase drives polar branch migration.

Bacteriophage T4↗

PubMed1995

Highly specific oxidative cross-linking of proteins mediated by a nickel-peptide complex.

The Ni(II) complex of the tripeptide NH2-Gly-Gly-His-COOH is shown to mediate efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid. Only proteins that associate specifically in solution are cross-linked under these conditions. Preliminary probes of the mechanism of the reaction suggest that the active intermediate may be a high-valent metal complex that attacks aromatic amino acids.

Acetates↗

PubMed1995

Homology dependence of UvsX protein-catalyzed joint molecule formation.

The bacteriophage UvsX protein is a "strand transferase" that promotes the pairing of homologous single and double-stranded DNAs. The efficiency of UvsX protein-mediated joint molecule formation between supercoiled duplex DNA and oligonucleotides is shown to have a sharp dependence on the degree of homology. The reaction proceeded efficiently with oligonucleotides containing 32 homologous positions but not with oligonucleotides containing only 24 homologous bases. This was shown to reflect an intrinsic homology requirement for the formation of stable joint molecules and was not caused by poor binding of the protein to short single-stranded DNAs. Even a single mismatch located in the middle of a region of 40 homologous nucleotides had a detectable effect on the efficiency of pairing. An in vitro recombinationally initiated DNA synthesis reaction that mimics the "secondary mode" of phage T4 DNA replication exhibited the same homology dependence.

Adenosine Triphosphatases↗

PubMed1995

From carpet bombing to cruise missiles: the 'second-order' mechanisms used by transcription factors to ensure specific DNA binding in vivo.

Transcription factors generally have only modest specificity for their target sites, yet must find them in a sea of non-specific DNA. Some transcription factors are expressed at very high levels, to ensure that, despite losses to non-specific binding, the promoter is still occupied (the carpet-bombing strategy). Others increase their binding specificity by collaborating with other factors in a variety of ways.

Animals↗

PubMed1995

The dangers of 'splicing and dicing': on the use of chimeric transcriptional activators in vitro.

Chimeric transcription factors composed of heterologous DNA-binding and activation domains are often used to study the regulation of gene expression. The fact that such preparations also contain molecules in which only one of the two domains is functional is often overlooked, but a surprisingly small proportion of inactive domains could cause serious problems in the interpretation of quantitative data.

DNA↗

PubMed1994

Strand exchange through a DNA-protein complex requires a DNA helicase.

The phage T4 uvsX and gene 32 proteins are capable of mediating homologous strand exchange, a central reaction in general genetic recombination, in vitro using naked DNA substrates. However, strand exchange is blocked by a sequence specific DNA-protein complex. Since protein-complexed substrates must be employed in vivo, this suggests that another factor(s) is required for strand exchange with protein-complexed DNAs. We show here that a DNA helicase, the T4 dda protein, allows the phage recombination machinery to drive branch migration through a RNA polymerase-promoter complex. This is the first observation of in vitro strand exchange using protein-bound substrates. These results suggest that a DNA helicase is a necessary component of the "protein machine" that mediates recombination in vivo.

Adenosine Triphosphatases↗

PubMed1994

Efficient unwinding of triplex DNA by a DNA helicase.

Sequence specific triple helix formation shows promise as a strategy for gene-specific inhibition of gene expression by blocking promoters or enhancers. Therefore, it is important to understand how this unusual structure affects DNA metabolic processes other than transcription. It has been shown that triplexes block in vitro DNA synthesis catalyzed by purified DNA polymerases. We report here that a purified DNA helicase unwinds a triple helical substrate with an efficiency similar to that observed with a comparable duplex species. These model studies suggest that triple helices will not seriously inhibit DNA replication or recombination in vivo, since DNA polymerases are preceded by helicases in the fully assembled replication holoenzyme.

Base Sequence↗

PubMed1994

Spectrophotometric assay for enzyme-mediated unwinding of double-stranded DNA.

A method is described for monitoring the enzyme-mediated unwinding of duplex DNA spectrophotometrically. The assay employs a partially duplex oligonucleotide substrate modified at the complementary end with coumarin and fluorescein moieties. When in close proximity the fluorescein quenches the fluorescence of coumarin. However, when the strands are separated by the action of a DNA helicase, the coumarin fluorescence increases greatly. Therefore, the progress of enzyme-mediated DNA unwinding can be measured in real time by fluorescence spectroscopy. This assay provides a simple method to screen for helicase inhibitors, which are of growing interest as potential anticancer agents. The application of this technique to kinetic analyses of the mechanism of action of DNA helicases is also discussed.

Base Sequence↗