Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding.

To investigate the mechanism of SWI/SNF action, we have analyzed the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes. We report here that the SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer. This disruption occurs without dissociating the DNA from the surface of the histone octamer. ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex. This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH. Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated. These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors.

High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex.

We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a "cylindrical clamp" effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes.

Estrogen receptor-induced DNA bending: orientation of the bend and replacement of an estrogen response element with an intrinsic DNA bending sequence.

The estrogen receptor (ER) binds to DNA fragments containing estrogen response elements (EREs) and causes them to bend. To characterize this ER-induced DNA bend and determine if it is involved in transcription activation, three different lines of investigation were used. Using MCF-7 human breast cancer cell extracts and circular permutation analysis, it was determined that molybdate-stabilized, unoccupied cytosolic ER was unable to bind to ERE-containing DNA fragments when maintained at 4 C, but that thermal activation enabled the cytosolic receptor to bind and bend ERE-containing DNA fragments to the same extent as ER present in whole cell extracts. DNA phasing analysis was utilized to determine that ER binding induced DNA fragments containing EREs to bend toward the major groove of the DNA helix. The orientation of this bend was the same with thermally activated, unoccupied cytosolic ER and with unoccupied ER, 17 beta-estradiol-occupied ER, and 4-hydroxytamoxifen-occupied ER present in whole cell extracts. Using transient cotransfection assays, the ability of an intrinsically bent DNA sequence to replace an ERE was tested. When a single consensus ERE, which is induced to bend 56 degrees on ER binding, was replaced with a 54 degrees intrinsic DNA bending sequence, transcription was effectively activated. Similar levels of transcription were also observed when promoters contained either a 108 degrees intrinsic DNA bending sequence or two consensus EREs. However, the 54 degrees DNA bending sequence and a single ERE were unable to cooperatively activate transcription. Because the magnitude and orientation of ER-induced DNA bends are the same with the unoccupied and occupied receptor, DNA bending alone probably does not function as a transcriptional switch to turn on gene transcription. However, DNA bending may be required to provide the architecture needed for modulation of target genes.

Structure and function of RecA-DNA complexes.

While the E. coli RecA protein has been the most intensively studied enzyme of homologous recombination, the unusual RecA-DNA filament has stood alone until very recently. It now appears that this protein is part of a universal family that spans all of biology, and the filament that is formed by the protein on DNA is a universal structure. With RecA's role in recombination given new and greatly increased significance, we focus in this review on the energetics of the RecA-mediated strand exchange and the relation between the energetics and recombination spanning heterologous inserts.

High-mobility-group 1 protein mediates DNA bending as determined by ring closures.

High-mobility-group 1 protein (HMG1) is an abundant eukaryotic DNA-binding protein, the cellular role of which remains ill-defined. To test the ability of HMG1 itself to mediate curvature in double-stranded DNA, we examined its effect on the phage T4 DNA ligase-dependent cyclization of short DNA fragments. HMG1 caused circle formation for fragments > or = 87 bp. Fragments of 123, 100, 92, and 87 bp did not cyclize in the absence of protein but formed covalently closed circular monomers efficiently in the presence of HMG1, indicating that the protein is capable of introducing bends into the duplex. The bending activity was maintained by a 79-amino acid polypeptide corresponding to a single HMG-box domain of HMG1. The binding affinity for the DNA minicircle was greater than for the corresponding linear fragment. These findings indicate that the role of HMG1 could involve both structure-specific recognition of prebent DNA and distortion of the DNA helix by bending and that the HMG-box domain may actually be responsible for this activity.

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

The synapsis event in the homologous pairing of DNAs: RecA recognizes and pairs less than one helical repeat of DNA.

A key step in homologous recombination is the alignment and pairing of homologous DNAs. The Escherichia coli RecA protein initiates pairing by binding to single-strand DNA, forming a helical nucleoprotein filament. We demonstrate that in the presence of the nonhydrolyzable ATP analogue adenosine 5'-[gamma-thio]triphosphate and ADP, RecA can pair a homologous oligonucleotide 15 bases long with a duplex DNA to yield synaptic complexes consisting of the oligonucleotide and duplex DNA stabilized by RecA. RecA can pair as few as eight bases of homology to form such synaptic complexes. The homologous DNAs remain paired to each other upon removal of RecA provided that the length of shared homology is at least 26 base pairs. Based on our findings and the work of others, we propose that in vitro, one helical turn of a RecA nucleoprotein filament containing approximately six RecA monomers and 15 bases of single-strand DNA is the functional unit sufficient to carry out the homology search.

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons.

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.

Involvement of topoisomerases in replication, transcription, and packaging of the linear adenovirus genome.

The role of topoisomerases in the replication of human adenovirus type 5 was investigated with topoisomerase inhibitors. Both topoisomerase I and topoisomerase II inhibitors blocked adenovirus replication when added at the time of infection. Both types of inhibitors induced strand cleavages at specific sites in the adenovirus early templates. The cleavage sites were mapped near the 5' and 3' ends of the genes transcribed early during infection. At late times after infection, camptothecin, a topoisomerase I inhibitor, inhibited adenovirus DNA replication and induced the formation of single- and double-stranded fragments with breakpoints located at defined regions of the viral genome. The topoisomerase II inhibitors, VP-16 (etoposide) and ellipticine, did not block adenovirus DNA replication and did not induce an appreciable amount of double-strand cleavages in the newly synthesized DNA. On the other hand, VP-16 promoted double-strand cleavages at specific sites of nonreplicating adenovirus DNA. The packaging of adenovirus DNA into virus particles, which contain supercoiled adenovirus DNA (M.-L. Wong and M.-T. Hsu, Nucleic Acids Res. 17:3535-3550, 1989), was inhibited by the topoisomerase II inhibitors. Transcription of adenovirus major late genes was inhibited by both topoisomerase I and topoisomerase II inhibitors. In addition, camptothecin caused a premature termination of major late transcription. Electron microscopic analysis showed that adenovirus templates late after infection were arranged in topologically constrained loop domains. Together, these data provide evidence for the requirement of topoisomerase activities in the replication, transcription, and packaging of the linear adenovirus genome.

Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy.

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.

Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative.