Alter DNA Topology Research Articles

Prebending the estrogen response element destabilizes binding of the estrogen receptor DNA binding domain.

Binding of many eukaryotic transcription regulatory proteins to their DNA recognition sequences results in conformational changes in DNA. To test the effect of altering DNA topology by prebending a transcription factor binding site, we examined the interaction of the estrogen receptor (ER) DNA binding domain (DBD) with prebent estrogen response elements (EREs). When the ERE in minicircle DNA was prebent toward the major groove, which is in the same direction as the ER-induced DNA bend, there was no significant effect on ER DBD binding relative to the linear counterparts. However, when the ERE was bent toward the minor groove, in a direction that opposes the ER-induced DNA bend, there was a four- to eightfold reduction in ER DBD binding. Since reduced binding was also observed with the ERE in nicked circles, the reduction in binding was not due to torsional force induced by binding of ER DBD to the prebent ERE in covalently closed minicircles. To determine the mechanism responsible for reduced binding to the prebent ERE, we examined the effect of prebending the ERE on the association and dissociation of the ER DBD. Binding of the ER DBD to ERE-containing minicircles was rapid when the EREs were prebent toward either the major or minor groove of the DNA (k(on) of 9.9 x 10(6) to 1.7 x 10(7) M(-1) s(-1)). Prebending the ERE toward the minor groove resulted in an increase in k(off) of four- to fivefold. Increased dissociation of the ER DBD from the ERE is, therefore, the major factor responsible for reduced binding of the ER DBD to an ERE prebent toward the minor groove. These data provide the first direct demonstration that the interaction of a eukaryotic transcription factor with its recognition sequence can be strongly influenced by altering DNA topology through prebending the DNA.

Bacterial diversity based on type II DNA topoisomerase genes.

Type II DNA topoisomerases are essential and ubiquitous DNA metabolic enzymes that alter DNA topology. Eubacteria have two indispensable type II DNA topoisomerases, DNA gyrase encoded by gyrB and gyrA and topoisomerase IV encoded by parE and parC. These genes belong to a single family whose members span both eukaryotes and prokaryotes. The highly conserved motifs in these genes provide a rationale for the design of universal primers used in the polymerase chain reaction in order to systematically generate a data set suitable for bacterial diversity studies at the macro-diversity level, as well as at the micro-diversity level displaying individual species and isolates. This family of genes is the subject of intensive biochemical and genetic analyses, which provide an opportunity for comprehensive understanding of sequence conservation and variability and their relationship to function. These genes are ideally suited for microbial identification and biodiversity analyses.

Cyclic AMP receptor protein positively controls gyrA transcription and alters DNA topology after nutritional upshift in Escherichia coli

The expression of a transcriptional gyrA-lacZ gene fusion throughout the Escherichia coli growth cycle and the effect that mutation delta crp39 had on this expression were studied. The data obtained indicate that the expression of gyrA is growth phase dependent and under the positive control of the cyclic AMP receptor protein (CRP). Complementation analysis of gyrA-lacZ expression with wild-type CRP or variant CRP pc (with a T-to-A mutation at position 158) in a CRP-deficient background suggests that this CRP action is mediated by a class I or class II CRP-dependent promoter(s). Our results also indicate that CRP may be involved in the modulation of DNA topology in the transition from the lag period to the exponential phase of growth.

Transcription, topoisomerases and recombination.

Transcription, DNA topoisomerases and genetic recombination are interrelated for several structural reasons. Transcription can affect DNA topology, resulting in effects on recombination. It can also affect the chromatin structure in which the DNA resides. Topoisomerases can affect DNA and/or chromatin structure influencing the recombination potential at a given site. Here we briefly review the extent to which homologous direct repeat recombination and site-specific recombination in eukaryotes are affected by transcription and topoisomerases. In some cases, transcription or the absence of topoisomerases have little or no effect on recombination. In others, they are important components in the recombinational process. The common denominator of any effects of transcription and topoisomerases on recombination is their shared role in altering DNA topology.

The chromatin-associated protein H-NS alters DNA topology in vitro.

H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.

Transcriptional activation of Mn-superoxide dismutase gene ( sodA) of Escherichia coli by MnCl 2

Transcription of the manganese-superoxide dismutase gene ( sodA) in Escherichia coli was shown to be activated by manganese. Addition of MnCl 2 increased the expression of β-galactosidase from a sodA::lacZ protein fusion and increased the concentration of mRNA transcribed from sodA + and sodA::lacZ constructs. The stimulatory affect of manganese on the expression of sodA::lacZ was greatly reduced (i.e., > 90%) in a strain harboring a fur mutation. We also found that manganese was capable of altering DNA topology. These results show that Mn 2+ causes activation of sodA transcription.

Topoisomerase inhibitors induce apoptosis in thymocytes

The effects of the inhibitors of topoisomerase I and II, camptothecin and etoposide, as well as novobiocin and adriamycin, on the DNA fragmentation and viability of mouse thymocytes in primary culture were examined. All inhibitors were shown to produce dose-dependent internucleosomal DNA cleavage by resolving isolated DNA by agarose-gel electrophoresis. The DNA fragmentation seemed to precede cell death, determined on the basis of LDH release, by a few hours. Etoposide-induced DNA fragmentation progressively increased after incubation and was enhanced by pretreatment with phorbol 12,13-dibutyrate, a phorbol ester capable of activating protein kinase C, whereas camptothecin-induced DNA fragmentation increased progressively after 12 h incubation and was unaffected by phorbol 12,13-dibutyrate-pretreatment. The process was also energy-dependent and required RNA and protein synthesis and protein phosphorylation, since it was inhibited by sodium azide, actinomycin D, cycloheximide and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine hydrochloride, a protein kinase inhibitor. DNA fragmentation was also inhibited by zinc ions, suggesting the involvement of a specific endonuclease in DNA cleavage. These phenomena are similar to those detected in thymocytes undergoing apoptosis following exposure to glucocorticoids (Cohen, J.J. and Duke, R.C. (1984) J. Immunol. 132, 38-42). Considering that topoisomerases function in cellular proliferation and differentiation by altering DNA topology, the results suggest that topoisomerases have important roles in T-lymphocyte ontogeny in the thymus and are in part involved in the elimination of autoreactive or harmful cells by an apoptotic process.

Estrogen receptor alters the topology of plasmid DNA containing estrogen responsive elements

We have recently used DNA containing estrogen responsive element (ERE) sequences for affinity purification to prepare calf uterine estrogen receptor (ER) at near homogeneity. The capacity of this purified ER to alter DNA topology upon binding was examined. Although the ER is not a topoisomerase, the presence of ER changes the distribution of topoisomers generated by incubation of plasmid DNA with excess wheat germ topoisomerase I. This effect is larger in plasmids containing a consensus ERE sequence. Two dimensional gel electrophoretic analysis suggested that interaction of ER and ERE causes negative supercoiling in regions of the plasmid accessible to topoisomerase I, resulting from overwinding of DNA contacting the ER. The extent of topological alteration was dependent on ER concentration. We suggest that the observed conformational changes in the DNA could have a role in regulation of transcription.

Mechanism of Tn3 resolvase recombination in vivo

To determine the physiologically important features of site-specific recombination by Escherichia coli Tn3 resolvase we analyzed the salient properties of the reaction in vivo. A two-plasmid system in which one plasmid served as substrate while the other encoded both resolvase and a thermolabile repressor of resolvase transcription provided controlled, synchronous recombination. Recombination proceeded rapidly and was promoted by (-) DNA supercoiling. The structures of the in vivo recombination products were predominantly the same as those previously identified in vitro. By examination of the products of successive rounds of recombination of a four-site substrate, we ruled out a tracking mechanism for site alignment. Inversion and plasmid fusion occurred in vivo at a much lower rate than resolution but ultimately reached a higher extent than found in vitro. We propose that inversion and fusion exploit topologically interlinked substrates that occur at low levels in vivo. This proposal is supported by the unexpected specificity of fusion. Our data imply that supercoiled DNA, the resolvase synaptic complex, and the mechanism of strand exchange are fundamentally similar in vivo and in vitro, but that the repertoire of resolvase substrates and products is expanded in vivo by the action of other enzymes that alter DNA topology.

Ribonucleoprotein and protein factors bind to an H-DNA-forming c-myc DNA element: possible regulators of the c-myc gene.

We have located a positive, cis-acting DNA sequence element within the 5' flanking DNA of the c-myc gene (-125 base pairs). This DNA sequence element has a large purine-pyrimidine strand asymmetry and can assume the H-DNA conformation. A factor with the properties of a ribonucleoprotein (RNP) interacts with this DNA region. The interaction of the c-myc DNA sequence element and the RNP involves an RNase H-sensitive mechanism and, therefore, may involve an RNA.DNA hybrid. In addition, a protein factor(s) binds to this DNA sequence element. DNA footprinting and mutant oligonucleotide binding/competition assays implicate a punctate, poly(G.C) recognition/binding sequence for the RNP factor, whereas the major protein factor requires two ACCCT sequence motifs for maximal binding. These results suggest that RNP and protein factors act as positive transcriptional regulators of the c-myc gene, perhaps by altering DNA topology.

An overlap between osmotic and anaerobic stress responses: a potential role for DNA supercoiling in the coordinate regulation of gene expression.

The regulation of several genes in response to osmotic and anaerobic stress has been examined. We have demonstrated a clear overlap between these two regulatory signals. Thus, the osmotically induced proU and ompC genes require anaerobic growth for optimum induction while the anaerobically induced tppB gene is also regulated by osmolarity. Furthermore, normal expression of tppB and ompC requires the positive regulatory protein OmpR, yet this requirement can be partially, or even fully, overcome by altering the growth conditions. Finally, the pleiotropic, anaerobic regulatory locus, oxrC, is also shown to affect expression of the osmotically regulated proU gene. The oxrC mutation is shown to affect the level of negative supercoiling of plasmid DNA and its effects on gene expression can be explained as secondary consequences of altered DNA topology. We suggest that there is a class of 'stress-regulated' genes that are regulated by a common mechanism in response to different environmental signals. Furthermore, our data are consistent with the notion that this regulatory overlap is mediated by changes in DNA supercoiling in response to these environmental stresses.

Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase.

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.

9] Escherichia coli phage T4 topoisomerase

Topoisomerases are enzymes that alter DNA topology by changing the linking number of circular duplex DNA molecules and by interconverting topologically knotted or catenated DNA forms. The so-called type II topoisomerases act by a mechanism involving the passage of a duplex segment of DNA through a transient double-strand break in another segment of DNA. A novel ATP-dependent type II topoisomerase with DNA-dependent ATPase activity is isolated from extracts of bacteriophage T4-infected E. coli cells. It has a high specific activity for topoisomerization reactions and can be easily purified to near homogeneity in milligram amounts. This chapter explains the interaction of T4 topoisomerase with DNA in the origin region of the T4 chromosome and attempts to reconstitute the initiation reaction in vitro in order to test this and other models for the involvement of the enzyme in the DNA replication process.