Scission of RNA by the chemical nuclease of 1,10-phenanthroline-copper ion: preference for single-stranded loops.

The scission of RNA by the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu) has been studied using a lac mRNA fragment and tRNAphe as substrates. Since the chemical mechanism of scission involves oxidative attack on the ribose, scission is observed at all nucleotides including dihydrouridine and Y-bases. Specificity for single-stranded loop regions is apparent from the similarity of the reactivity of OP-Cu to the single-strand specific reagents dimethyl sulfate and diethyl pyrocarbonate using the fragment of lac mRNA as a substrate. Similar preference is observed in the reaction with tRNA although scission in the helical acceptor stem is also observed.     

DNA cleavage in chromatin spacer segments by a non-enzymic probe, 1,10-phenanthroline-copper complex

The action of 1,10-phenanthroline-copper complex under aerobic conditions in the presence of 2-mercaptoethanol on chromatin DNA in murine thymocyte nuclei was studied. At limited oxygen supply primarily multiple single-strand breaks of DNA in the spacer segments are observed while the core DNA segments in chromatin remain intact. After the single-strand breaks accumulate in both DNA strands (under conditions of improved oxygen supply), double-strand cleavage of DNA to fragments of nucleosomal size becomes apparent. This regular character of DNA degradation in chromatin is apparently due to the preferential binding of the 1,10-phenanthroline-copper complex to DNA of the spacer segments and to the localized generation of damaging radicals.Abbreviations OP 1,10-phenanthroline - OP-Cu 1,10-phenanthroline-copper complex - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulphate - 2-ME 2-mercaptoethanol     

Drosophila engrailed-1,10-phenanthroline chimeras as probes of homeodomain-DNA complexes.

We have converted the Drosophila engrailed homeodomain into a sequence-specific nuclease by linking the protein to the chemical nuclease 1,10-phenanthroline-copper (OP-Cu). Unique cysteines were introduced at six positions into the homeodomain by site-directed mutagenesis for the covalent attachment of OP-Cu. The varied DNA-binding affinity and specificity of these mutants and the DNA cleavage pattern of their OP-Cu derivatives allowed us to assess the crystal structure of the engrailed homeodomain-DNA complex. We have also achieved site-specific double-stranded DNA scission with one of the homeodomain mutants, E28C, which has the potential of being used to identify engrailed binding sites in the genome. Because the homeodomain is so well conserved among members of the homeodomain-containing protein family, other homeodomain proteins can be converted into nucleases by attaching OP-Cu at position 28 of their homeodomains.     

Sequence specificity of the deoxyribonuclease activity of 1,10-phenanthroline-copper ion

A statistical analysis of a data set composed of over 1600 scission events of DNA produced by the 2:1 1,10-phenanthroline-copper complex (OP-Cu) has demonstrated that the nucleotide 5' to the site of phosphodiester bond scission is a primary influence in the kinetics of cleavage at any sequence position. The scission was less affected by the 3' neighbor. For each of the sixteen possible dinucleotides, a kinetic parameter can be computed reflecting scission at the 3' nucleotide. When used to predict the scission pattern of a DNA sequence not part of the present data set, correlation coefficients of about 0.6 between predicted and observed patterns were obtained.     

Variability of the intracellular ionic environment of Escherichia coli. Differences between in vitro and in vivo effects of ion concentrations on protein-DNA interactions and gene expression

Effects of changes in intracellular ion concentrations on the interactions of Escherichia coli lac repressor with lac operator mutants and on the interactions of RNA polymerase with various promoters have been investigated in vivo. The intracellular ionic environment was reproducibly varied by changing the osmolality of the 4-morpholinepropanesulfonic acid minimal growth medium. As the osmolality of the growth medium is varied from 0.1 to 1.1 osmolal, the total intracellular concentration of K+ increases linearly from 0.23 +/- 0.03 to 0.93 +/- 0.05 molal and the total intracellular concentration of glutamate increases linearly from 0.03 +/- 0.01 to 0.26 +/- 0.02 molal. The sum of the changes in the total concentrations of these two ions appears sufficient to compensate for a given change in external osmolality, indicating that K+ and glutamate are the primary ionic osmolytes under these conditions and that these ions are free in the cytoplasm. In support of this, in vivo 39K NMR experiments as a function of external osmolality indicate that changes in the total cytoplasmic K+ concentration correspond to changes in the free cytoplasmic K+ concentration. Extents of interaction of lac repressor and RNA polymerase with their specific DNA sites were monitored by measuring the amounts of beta-galactosidase produced under the control of these sites. For both lac repressor and RNA polymerase, it was found that formation of functional protein-DNA complexes in vivo is only weakly (if at all) dependent on intracellular ion concentration. These results contrast strongly with those obtained on these systems in vitro, which showed that both the equilibria and kinetics of binding are extremely salt-dependent. We discuss several possible mechanisms by which E. coli may compensate for the potentially disruptive effects of these large changes in the intracellular ionic environment.     

Interlocking of plasmid DNAs due to Lac repressor-operator interaction.

The presence of a single lac repressor binding sequence on plasmid DNAs is shown to mediate the formation of interlocked dimers in E. coli. The presence of both homo- and hetero-interlocked dimers suggests that the lac repressor complex can bring together randomly two plasmid DNA molecules to facilitate gyrase-mediated interlocking. The exclusive formation of multiply intertwined dimers also suggest that the lac repressor complex may bind simultaneously to a pair of replicated daughter plasmid molecules prior to their segregation. The formation of interlocked plasmid DNAs can be indicative of interaction between two DNA bound proteins in vivo.     

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

Escherichia coli lac repressor is a tetrameric protein composed of 360 amino acid subunits. Considerable attention has focused on its N-terminal region which is isolated by cleavage with proteases yielding N-terminal fragments of 51 to 59 amino acid residues. Because these short peptide fragments bind operator DNA, they have been extensively examined in nuclear magnetic resonance structural studies. Longer N-terminal peptide fragments that bind DNA cannot be obtained enzymatically. To extend structural studies and simultaneously verify proper folding in vivo, the DNA sequence encoding longer N-terminal fragments were cloned into a vector system with the coliphage T7 RNA polymerase/promoter. In addition to the wild-type lacI gene sequence, single amino acid substitutions were generated at positions 3 (Pro3----Tyr) and 61 (Ser61----Leu) as well as the double substitution in a 64 amino acid N-terminal fragment. These mutations were chosen because they increase the DNA binding affinity of the intact lac repressor by a factor of 10(2) to 10(4). The expression of these lac repressor fragments in the cell was verified by radioimmunoassays. Both wild-type and mutant lac repressor N termini bound operator DNA as judged by reduced beta-galactosidase synthesis and methylation protection in vivo. These observations also resolve a contradiction in the literature as to the location of the operator-specific, inducer-dependent DNA binding domain.     

Analysis of the gel electrophoresis of looped protein-DNA complexes by computer simulation

The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis of a numerical model that has been applied to systems such as lac repressor-lac operator DNA, in which a protein binds in two different modes to linear DNA carrying two specific binding sites. Three complexes may be formed: (1) a linear 1:1 complex with one protein molecule bound to one site on the DNA molecule; (2) a 1:1 complex in which a single protein molecule is bound to both sites simultaneously, thereby inducing a large DNA loop; and (3) a 2:1 linear complex in which two protein molecules are bound in tandem, each occupying a single site. The computational model affords a quantitative numerical simulation of the observed gel electrophoretic patterns produced by titration of the DNA with protein and provides new insights into the shape and nature of the patterns. In particular, the patterns may represent unimodal or bimodal reaction zones. Nevertheless, analysis of the peaks in the patterns obtained at low DNA and high protein concentration provides essential information as to the stoichiometry of the complexes and satisfactory estimates of association constants. The theory thus provides the experimenter with guidelines for quantitative evaluation of the results of gel retardation assays of the particular system under investigation, once protein-induced DNA (or RNA) loops have been established by independent physical or chemical methods. It is suggested that these insights might also find application to systems involving the binding of two or three different proteins to DNA with loop formation.     

Radiolysis of lac repressor by gamma-rays and heavy ions: a two-hit model for protein inactivation

Upon gamma-ray or argon ion irradiation of the lac repressor protein, its peptide chain is cleaved and the protein loses its lac operator-binding activity, as shown respectively by polyacrylamide gel electrophoresis and retardation gel electrophoresis. We developed phenomenological models that satisfactorily account for the experimental results: the peptide chain cleavage model considers that the average number of chain breaks per protomer is proportional to the irradiation dose and that the distribution of the number of breaks per protomer obeys Poisson's law. The repressor inactivation model takes into account the quaternary structure (a dimer of dimer) and the organization of the repressor in domains (two DNA binding sites, one per dimer). A protomer is inactivated by at least two different radiation-induced damages. A dimer is inactivated when at least one of the two protomers is inactivated. A tetramer is inactivated when both dimers are inactivated. From the combination of both models, we can deduce that chain cleavage cannot account for the protein inactivation, which should mainly result from oxidation of amino acid side chains. Indeed, particularly oxidizable and accessible amino acids (Tyr, His) are involved in the DNA binding process.