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The high-mobility group (HMG) protein A2 has been studied mostly in the mouse where its function seems critical for embryonic cell growth and adipogenesis, leading to a pygmy phenotype with greatly reduced fat tissue in homozygous knock out mice. We showed recently that among the major HMG proteins, HMGA2 is highly expressed in two human embryonic stem (hES) cell lines. Here, we employed siRNA technology in combination with quantitative reverse transcriptase polymerase chain reaction, stem cell-specific microarray analyses, and cell proliferation assays in order to probe into HMGA2's role(s) in pluripotent hES cells. Our results establish HMGA2 as a regulator of human genes linked to mesenchymal cell differentiation, adipogenesis, and hES cell growth.

In this work, we performed a systematic study of perfect and nonspacer palindromes present in human genomic DNA, and we investigated palindrome distribution over the entire human genome and over the functional regions such as the exon, intron, intergenic, and upstream regions (2,000 bp upstream from translational start site). We found that 24 palindrome-abundant intervals are mostly located on G-bands, which condense early, replicate late, and are relatively A+T rich. In general, palindromes are overrepresented in introns but underrepresented in exons. Upstream region has enriched palindrome distribution, where palindromes can serve as transcription factor binding sites. We created a Human DNA Palindrome Database (HPALDB) which is accessible at http://vhp.ntu.edu.sg/hpaldb . It contains 12,556,994 entries covering all palindromes in the human genome longer than 6 bp. Queries can be performed in different ways. Each entry in the database is linked to its location on NCBI's human chromosome Map Viewer.

Architectural proteins that reconfigure the paths of DNA segments are required for the establishment of functional interfaces in many genomic transactions. A single-chain derivative of the DNA architectural protein integration host factor was found to adopt two stable conformational states in complex with a specific DNA target. In the so-called open state, the degree of protein-induced DNA bending is reduced significantly compared with the closed state. The conformational switch between these states is controlled by divalent metal binding in two electronegative zones arising from the lysine-to-glutamate substitution in the protein body proximal to the phosphate backbone of one DNA arm. We show that this switch can be employed to control the efficiency of site-specific recombination catalyzed by lambda integrase. Introduction of acidic residues at the protein-DNA interface holds potential for the design of metal-mediated switches for the investigation of functional relationships.

Undifferentiated transcription factor 1 (UTF1) was identified first in mouse embryonic stem cells and is also expressed in human embryonic and adult stem cells. UTF1 transcription ceases at the onset of differentiation, which clearly distinguishes it from less sensitive pluripotency markers, such as Oct4 or Nanog. We present here two transgenic hESC lines, named ZUN. Each line harbors one copy of the UTF1 promoter/enhancer driving a resistance gene and yielded highly homogeneous cultures under selection pressure, with a larger proportion of Oct4 and Sox2 positive cells. While ZUN cultures, like parental HUES8 cultures, retained the capacity to differentiate into tissues of all three germ layers using a SICD mouse teratoma model, they surprisingly exhibited an increased refractoriness to various differentiation cues in vitro. Together with its small size of only 2.4 kb for the entire cassette, these features render our selection system a powerful novel tool for many stem cell applications and human somatic cell reprogramming strategies.

The state of chromatin in human embryonic stem (hES) cells is a key factor determining stem cell identity. The non-histone chromatin-associated factor HMGA2 has been studied mostly in the mouse where its function seems critical for embryonic cell growth and adipocytic cell differentiation. Here we show that HMGA2 is highly expressed in two undifferentiated human embryonic stem cell lines at a level of at least 10(5) copies per individual stem cell. Interestingly, expression is further upregulated by a factor of three at day 7 of embryoid body formation, before it quickly drops to or below the level found in undifferentiated cells. We also show that HMGA2 is stably associated with inter- and metaphase hES cell chromatin, and that up to 12 HMGA2 protomers stably associate in vitro with a single nucleosome core particle of known atomic structure. Our data lend support to the possibility that HMGA2 interacts with nucleosomes in a way that imposes a global effect on the state of ES cell chromatin, which may contribute to the establishment of both ES cell identity and the initiation of specific differentiation programs.

The great potential of human embryonic stem cells (hESCs) in basic research, regenerative medicine, and gene therapy is widely recognized. Controlled manipulation of hESC genomes through sequence-specific DNA recombination (SSR) may play a significant role in future hESC applications. However, very little is known about the functionality of SSR systems in hESCs. We demonstrate here that mutant phage lambda integrase, phage P1 Cre recombinase, and mutant gammadelta resolvase displayed distinct activities on episomal recombination substrates. Interestingly, cofactor-independent lambda integrase catalyzed the integrative pathway five times more efficiently than the excisive pathway. Such a degree of directionality in hESCs could be explored for sequential gene insertions into predetermined genomic sequences. We also report an improved, easy-to-use plasmid transfection system that employs silica microspheres and, in combination with SSR, could be applied to hESC genome engineering.

Integration host factor (IHF) is a heterodimeric, site-specific DNA-binding and DNA-bending protein from Escherichia coli. It is involved in high-precision DNA transactions where it serves as a key architectural component of specialized nucleoprotein structures (snups). We described recently a novel approach for protein engineering using a single polypeptide chain IHF, termed scIHF2, as a first example. ScIHF2 is made up of the alpha subunit of IHF which was inserted into the beta subunit at peptide bond Q39/G40 via two short linkers. The monomer behaves very similarly to the heterodimeric, parental IHF in biochemical and functional assays. Here, we describe an extension of this approach in which we shortened either one or both linkers by one amino acid, thereby generating three new variants termed scIHF1, 3, and 4. These variants exhibit distinct DNA-binding properties, different phenotypes in site-specific integrative and excisive recombination by phage lambda integrase in vitro, as well as in pSC101 replication assays in a DeltaIHF E. coli host. We also introduced a K45E substitution within the alpha domain of scIHF3 and based on electrophoretic mobility shift assays (EMSAs), argue that it significantly changes the DNA trajectory within the protein-DNA complex. Our results indicate that IHF's pleiotropic roles in DNA transactions inside E. coli require different types of high-precision DNA architectural activities. The scIHF variants described here will help to explore further how flexible these requirements are.

The heterodimeric integration host factor (IHF) is a site-specific DNA-binding and DNA-bending protein from Escherichia coli. It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication. IHF's ability for concerted binding and bending of DNA is key to its biological function. Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire alpha-subunit of IHF into the beta-subunit. DNA binding and DNA bending assays revealed that purified wild-type IHF and scIHF2 behave very similarly. Further, scIHF2 is required for site-specific integrative recombination by phage lambda integrase and for pSC101 replication in a DeltaIHF E.coli host. It also triggers site-specific integrative and excisive recombination in vitro to the same extent as the wild-type protein. We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase. Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or benefit from sequence-specific high precision DNA bending.

Mutant lambda integrases catalyze site-specific DNA recombination in the absence of accessory factors IHF, XIS, and negative DNA supercoiling. Here we investigate the effects that a human cellular environment exerts on these reactions in order to (i) gain further insights into mechanistic aspects of recombination in eukaryotic cells and (ii) to further develop the Int system for biotechnological applications. First, we compared intra- and intermolecular integrative as well as excisive recombination pathways on episomal substrates after co-transfection with recombinase expression vectors. Our results demonstrate that, within 24 hours after transfection, intermolecular recombination by mutant integrase is at least as efficient as intramolecular recombination. Second, a significant intermolecular recombination activity was observed between two copies of a recombination site containing only the 21 bp comprising core-type DNA sequence. This basic activity was stimulated several-fold when arm-type DNA sequences were present in addition to core sites. Therefore, one recombination pathway in human cells involves mutant integrases bound solely at core sites, which is reminiscent of the Flp/FRT and Cre/loxP pathways. The stimulatory effect of arm-type sequences could be explained by an increase in integrase concentration in the vicinity of core sites. We show, in addition, that an N-terminal truncated mutant integrase exhibited only a very weak recombinogenic activity in a eukaryotic background. This result strengthens a functional role for the N-terminal domain in recombination in addition to its arm-type DNA-binding activity. Finally, we demonstrate that low level integrative recombination by wild-type integrase is stimulated when purified integration host factor is co-transfected. This corroborates our previous conclusion that sufficient amounts of eukaryotic protein co-factors, which could functionally replace IHF, are not present in human cells. It also provides a potential means to control site-specific recombination in eukaryotic cells., (Copyright 2002 Elsevier Science Ltd.)

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols., (Copyright 2002 Wiley-Liss, Inc.)

DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures. Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci. The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length. However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired. This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases. Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin. Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins. The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin.

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled., (Copyright 2001 Academic Press.)

The speed of chemical reactions is proportional to the concentration of molecules involved. Since proteins catalyze most of the essential reactions inside a living cell, their concentration should be as high as possible. An economical way to achieve this is through the establishment of small cell compartments. We propose that within these compartments, two types of local concentration effects are at work. (1) With local concentration type I reactions, multimeric proteins bound to a specific DNA sequence have an increased local concentration for a second DNA site sufficiently close-by, or for proteins bound to such a site. (2) For type II effects, DNA can be used as a scaffold to build unique nucleoprotein complexes that would otherwise not exist free in solution. These complexes are proficient in establishing longer-range interactions with similarly unique complexes located far away on the genome. We discuss the consequences of these local concentration effects in the light of the markedly different sizes of prokaryotic and eukaryotic cells and of their genomes.

We have transferred the prokaryotic gammadelta resolvase system to mammalian cells and present a comparative analysis of recombination by wild-type and two mutant resolvases (E124Q and E102Y/E124Q). Transient co-transfection assays using beta-galactosidase as reporter for recombination reveal that episomal DNA does not contain a significant level of unconstrained negative supercoiling, since only mutant resolvases are recombination-proficient. We also show that the efficiency of recombination by the resolvase double mutant is comparable to that observed with Cre, which indicates that resolvase can be used as a new tool for controlled manipulations of episomal DNAs.

DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively. Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange. Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase. The concomitant binding of Gin to sites I and III in res, however, inhibits recombination. A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency. Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange. Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding., (Copyright 2000 Academic Press.)

Phage lambda integrative and excisive recombination normally proceeds by a pair of sequential strand exchanges. During the first exchange reaction, the "top" strand in each recombination site is cleaved, exchanged, and religated generating a Holliday junction intermediate. This intermediate DNA structure is resolved through a pair of reciprocal "bottom" strand exchanges, leading to recombinant products. The strict co-ordination of exchange reactions ensures religation between correct partner strands only. Here we show that the directionality of recombination is altered in vivo by two mutant integrases, Int-h (E174 K) and a double mutant Int-h/218 (E174 K/E218 K). This change in directionality leads to deletion instead of inversion on substrates that carry inverted attachment sites and, depending on the pair of target sites employed, requires the presence or absence of integration host factor. Neither Fis nor Xis is involved in deletion. Sequence analyses of deletion products reveal that the newly generated hybrid attachment site exhibits a reversed genetic polarity. We demonstrate that only one of two possible hybrid site configurations is generated and discuss two pathways leading to deletion. In the first, deletion results from a wrong alignment of the two recombination sites within the synaptic complex. In the second pathway, the unco-ordinated cleavage by the mutant integrases of all four DNA strands present in a conventional Holliday junction intermediate leads to two double-stranded breaks, whereby the subsequent rejoining between "wrong" partner strands appears restricted to only two strands., (Copyright 1999 Academic Press.)

The presence of intervening sequences or introns in eukaryotic genes has been known for more than 20 years, and the mechanisms underlying RNA splicing have been studied in depth both genetically and biochemically. In recent years, however, an increasing number of bacterial genes have been introduced into higher eukaryotes as important tools for genetic studies. Their gene products are frequently used as an indirect measure for cell type-specific promoter activity, as, for example, in the case of chloramphenicol acetyl transferase (CAT assay) or beta-galactosidase. Here we show that RNA splicing of two prokaryotic genes encoding site-specific DNA recombinases occurs in eukaryotic cells. In one case, splicing is only observed after treatment of cells with the cytokine alpha interferon. We further demonstrate that mutating an intragenic donor splice site in a bacterial gene apparently activates a second, alternative splicing pathway. In conjunction with previous reports, our findings should also be regarded as a warning that splicing of bacterial genes in higher eukaryotes is a more common phenomenon than presently recognized, which may be difficult to overcome and may cause problems in the interpretation of experimental results.

Background: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon-encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites., Results: The resolvase encoded by transposable element ISXc5 from the gram-negative phytopathogen Xanthomonas campestris shows two features which distinguish it from other known resolvases. First, it is more closely phylogenetically related to invertases than other resolvases. In particular, two functionally important regions seem highly conserved between this resolvase and members of the invertase subfamily. Second, the enzyme exhibits a large extension of its carboxy-terminal domain with unknown function. We purified ISXc5 resolvase and analysed its resolution reaction in vitro. Our biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by gammadelta resolvase. However, despite its apparent similarity to invertases, we were unable to detect recombination on standard substrates for DNA inversion, in either the presence or absence of Fis., Conclusions: ISXc5 resolvase employs a reaction mechanism which is common to members of the resolvase family. Its position near the evolutionary borderline to invertases and its high degree of identity within two functionally important regions with members of the DNA invertase subfamily suggest that only a few replacements of critical residues may suffice to convert this resolvase into a functional, possibly Fis-dependent invertase.

The translocation of a transcription complex can transiently introduce positive and negative superhelical windings into the template DNA. To gain further insight into this dynamic DNA supercoiling mechanism and its possible involvement in biological processes, we employed an in vitro system in which site-specific recombination by gammadelta resolvase is topologically coupled to transcription-induced negative supercoiling. Our kinetic experiments suggest that recombination is closely linked to the process of supercoiling by transcription. We utilized the known high speed at which two resolvase-bound recombination sites can pair to form a synaptic complex in kinetic experiments with DNA substrates containing three recombination sites. Our data provide evidence for the existence of a transient gradient of negative supercoiling. Such a gradient seems to be predominantly a consequence of DNA double helix rotation behind a translocating RNA polymerase and originates within a broad region up to two kilobase-pairs upstream of the transcriptional start site. We further demonstrate that the topological coupling between transcription and recombination is not affected when the DNA-bending protein integration host factor from E. coli is bound to multiple sites within the phage lambda attachment region. We discuss implications of our in vitro findings with respect to possible in vivo functions of the dynamic nature of transcription-induced supercoiling.

The global superhelical state of intracellular DNA is stringently controlled by topoisomerase action. Little is know, however, about topoisomerase-directed relaxation of localized DNA supercoiling generated by protein tracking processes such as transcription. Here we use transcription by a yeast Gal4 and phage T7 RNA polymerase fusion protein to induce localized supercoiling which, in turn, triggers site-specific DNA recombination by gamma delta resolvase. We demonstrate that only large amounts of eukaryotic topoisomerase I interfere, through supercoiling relaxation, with the topological coupling between transcription and recombination. The additional presence of a strong cleavage site for topoisomerase I has little influence on the relaxation of localized supercoiling. We also show that high levels of human topoisomerase II fail to compete with transcription-driven recombination. However, drastically reduced amounts of either enzyme completely suppress recombination of overall supercoiled DNA. Together, our results reveal a marked difference in topoisomerase requirement to relax transcription-induced and global DNA supercoiling. We discuss possible reasons for this difference and conclude that localized supercoiling frequently may escape relaxation by eukaryotic topoisomerases to mediate topological couplings between DNA transactions.

An interplay between DNA-dependent biological processes appears to be crucial for cell viability. At the molecular level, this interplay relies heavily on the communication between DNA-bound proteins, which can be facilitated and controlled by the dynamic structure of double-stranded DNA. Hence, DNA structural alterations are recognized as potential tools to transfer biological information over some distance within a genome. Until recently, however, direct evidence for DNA structural information as a mediator between cellular processes was lacking. This changed when the concept of transient waves of DNA supercoiling, induced by proteins tracking along the right-handed DNA double helix, came into the limelight. Indeed, a number of observations now suggest that helix tracking-induced DNA structural information might be exploited to participate in the regulation of a variety of DNA transactions in vivo.

Transcription of a topologically relaxed, circular DNA triggers recombination between two directly repeated res sites by gamma delta resolvase in vitro. This activation of recombination depends on the res site-to-site distance and the orientation of sites with respect to the direction of RNA polymerase tracking. In addition to functioning as a site-specific recombinase, gamma delta resolvase acts as a site-specific topoisomerase and increases the topological linking number of templates during transcription. The data suggest that the link between transcription and recombination could be negative DNA supercoiling that transiently builds up on a relatively short DNA segment in the wake of an advancing RNA polymerase. Surprisingly, transcription-driven recombination is not inhibited by the presence of large amounts of eukaryotic topoisomerase type I, indicating that site-specific recombination can override relaxation by diffusible topoisomerases. This in vitro system might therefore serve as a model for some transcription-directed recombination events observed in vivo.

Fast and efficient recombination catalyzed by gamma delta resolvase in vitro requires negative DNA supercoiling of plasmid substrates. The current model for recombination suggests that supercoiling is required to drive DNA strand exchange within a synaptic complex by 'simple rotation' of DNA-linked resolvase promoters. Surprisingly, DNA knots are recombined efficiently in the absence of supercoiling, whereby the rate of recombination increases with the number of irreducible DNA segment crossings, or nodes, within each substrate knot. Recombination products contain three knot nodes less than substrates, suggesting that a reduction in writhe drives the reaction. However, the proposed protomer rotation model predicts that writhe is not altered during the process of strand transfer but, instead, is reduced only when a synaptic complex disassembles after strand exchange. I present evidence that recombination of knotted and of linear substrates coincides with a disassembly of synaptic complexes. The results lead to a variant model for strand exchange on non-supercoiled substrates in which a specific disassembly of the synaptic complex, triggered by a reduction in writhe, guides the cleaved DNA into the recombinant configuration.

We investigated the effect of left-handed Z-DNA on transcription by bacteriophage T7 RNA polymerase in vitro and, surprisingly, found that the enzyme can efficiently utilize a template containing a stretch of left-handed DNA close to the promoter. Analysis of transcription products revealed that only a small fraction of elongating polymerases abort transcription either at the promoter proximal or at the distal B-to-Z junction and, even less frequently, within the stretch of left-handed DNA. Our results indicate that, unlike E. coli RNA polymerase, T7 RNA polymerase can utilize a template with a CG stretch in an alternate conformation. In contrast, polymerases are completely blocked at the promoter proximal junction by a monoclonal antibody directed against Z-DNA. This blockage remains stable over a remarkable time, even when negative supercoiling is released by linearization of the template. Together with our recent finding of transcription-induced formation of Z-DNA (3), our data provide an example for a possible auto-regulatory mechanism that employs a change in DNA conformation.

We have tested in vitro the occurrence of a B-to-Z transition in a region of alternating purines and pyrimidines as a consequence of transcription-induced negative supercoiling. By using a monoclonal antibody as a specific Z-DNA stabilizing agent, we demonstrate that the formation of left-handed DNA can transiently occur when a topologically unconstrained template is transcribed. The B-to-Z transition, observed in a subpopulation of templates, appears to be induced by negative supercoiling generated in the wake of an elongating T7 RNA polymerase. Consistent with this, the presence of topoisomerases during the transcription period prevents the change in DNA conformation. These data agree with the 'twin-supercoiled-domain' model for transcription of Liu and Wang (1). Interestingly, our results suggest that the diffusion rate of transcription-induced superhelical twists must be relatively slow compared to their generation, and that under in vitro conditions localized transient supercoiling can reach unexpectedly high levels.

During site-specific recombination by the gamma delta resolvase, four DNA strands are broken, exchanged, and religated. This exchange is carried out within a DNA-protein complex, the synaptosome, in which the recombination sites, res, are aligned. The domain of resolvase that binds to a res site is distinct from the domain that breaks and rejoins the DNA. We tested whether the catalytic domain acts on the res site to which its binding domain is bound (in cis) or on the opposing res site in the synaptic complex (in trans). We constructed a hybrid synaptosome in which one res site is bound to wild-type resolvase and the other is bound to a mutant resolvase that binds normally but is unable to break DNA. From the pattern of strand breakage in the reaction intermediate containing resolvase covalently attached to DNA, we conclude that resolvase attacks predominantly, if not exclusively, in cis. Because cis breakage and reunion per se cannot lead to recombination, our results support a model in which DNA exchange is guided by an exchange of resolvase subunits between the breakage and reunion events.

The nitrogen regulatory protein NtrC of enteric bacteria activates transcription of the glnA gene by catalyzing isomerization of closed complexes between RNA polymerase and the glnA promoter to open complexes. NtrC binds to sites upstream of glnA that have properties of eukaryotic transcriptional enhancers. NtrC-binding sites were found to facilitate open complex formation when these sites and the glnA promoter were located on different rings of a singly linked catenane, but not when the two rings were decatenated. The results provide evidence that NtrC contacts RNA polymerase-promoter complexes in a process mediated by formation of a DNA loop. NtrC-binding sites serve to tether NtrC near the glnA promoter, thereby increasing the frequency of collisions between NtrC and polymerase-promoter complexes.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.

Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity.

Highly torsionally stressed replicative intermediate SV40 DNA molecules are produced when ongoing replicative DNA synthesis is inhibited by aphidicolin, a specific inhibitor of DNA polymerase alpha. The high negative superhelical density of these molecules can be partially released by intercalating drugs such as chloroquine or ethidium bromide. The torsionally stressed replicative intermediates bind to monoclonal anti-Z-DNA antibodies. Electron microscopy of anti-Z-DNA cross-linked to torsionally stressed replicative intermediates shows that the antibody specifically binds close to the replication forks. The superhelical structures are not formed when SV40 DNA replication is inhibited by both aphidicolin and novobiocin, suggesting that a topoisomerase type II-like enzyme is somehow involved in the introduction of torsional strain in replicative intermediate DNA. One interpretation of our data is that fork movement continues to some rather limited extent when SV40 DNA synthesis in replicative chromatin is blocked by aphidicolin. After deproteinization, the exposed single-stranded DNA branches reassociate to form paranemic DNA structures with left-handed helical stretches, while the reduced linking number of the parental strands induces a high negative superhelical density.

A key question concerning the mechanism of transcriptional activation by enhancers is about the role of the DNA that connects the enhancer to the promoter. The linking DNA will be important if a regulatory protein(s) binds to the enhancer and then tracks or slides along the DNA to the promoter, or if, on binding, the protein(s) alters the topological state of the DNA. By contrast, if the linking DNA loops out to allow the formation of a promoter-enhancer complex, or if the enhancer increases the local concentration of a transcription factor, co-linearity of the promoter and the enhancer will not be strictly required. In Xenopus laevis, the transcription of the ribosomal RNA genes is stimulated by an enhancer composed of repetitive sequences in the intergenic spacer regions. These repetitive elements contain 60 or 81 base pairs, and their activity is relatively independent of their position and orientation. When the enhancer and promoter sequences are each located on separate DNA molecules, however, the enhancer is no longer able to augment transcription. We have now tested whether or not this apparent requirement for having the enhancer and promoter in cis can be overcome by keeping them in close proximity while locating them separately on different molecules. This was achieved by generating multiply intertwined, dimeric-catenanes in which the enhancer and promoter were located in trans on different rings. By injecting these catenanes into frog oocytes and measuring the activity of the enhancers in a series of competition assays, we were able to demonstrate that such enhancers can augment transcription in vivo.

Various monoclonal antibodies specific for simian virus 40 large tumor antigen (T antigen) inhibit the elongation process of viral DNA replication in an in vitro system. The results provide strong evidence for a function intrinsic to T antigen during ongoing replicative-chain elongation. The antibody inhibition studies were further used to establish a correlation between the known biochemical activities of T antigen and its function during the elongation phase. The data demonstrate that, in addition to DNA binding and ATPase, a third function of T antigen is required for replicative chain elongation. This function is most probably related to the recently described DNA helicase activity of T antigen. This conclusion is based on the following results: aphidicolin treatment of actively replicating simian virus 40 minichromosomes causes a partial uncoupling of parental DNA strand separation and DNA synthesis; the strand separation reaction is blocked by the same monoclonal antibodies which strongly inhibit the elongation process. DNA helicase activity of isolated T antigen is equally well inhibited by the same set of monoclonal antibodies that affect minichromosome replication in vitro.

We studied the site orientation specificity for recombination by purified Tn3 resolvase. With standard plasmid substrates, resolvase acts only on directly repeated recombination sites. Knotting, however, makes inverted site substrates equally efficient. The structure of the knotted products of recombination shows that the DNA wrapped around resolvase in the synaptic intermediate has the same local geometry as the standard substrate but is reversed in topological sign. Similarly, the same strand exchange with the two substrates generates supercoils with opposite signs. Thus, DNA geometry rather than topology is critical for these features of recombination. The knotted inverse substrate like the direct site substrate must be (-) supercoiled under standard reaction conditions. However, under conditions in which supercoiling is not required, the structure of the knotted product is apparently the same. This indicates that the unique direction of strand exchange is determined by the structure of the synaptosome and not by (-) supercoiling of the substrate.