Marc Leng

GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming.

The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)n sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3′ terminus and its re‐synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB‐induced ‘cleavage‐and‐restart’ process to become catalytically competent. Clearly, by promoting transcript shortening and re‐alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.

Solvation of the left-handed hexamer d(5BrC-G-5Brc-G-5BrC-G) in crystals grown at two temperatures

Crystals of the hexadeoxyoligomer d(5BrC-G-5BrC-G-5BrC-G) were grown at different temperatures (5 degrees C, 18 degrees C and 37 degrees C) in the absence of divalent cations. The crystals grown at 5 degrees C did not diffract X-rays, while those grown at 18 degrees C and 37 degrees C did. The oligomer adopts the left-handed ZI conformation in both crystals. The main difference resides in a more extensive hydration shell in the crystal grown at high temperature than in the crystal grown at low temperature. The high-temperature crystal displays a spine of hydration running deep in the minor groove and linking exocyclic O-2 atoms of the pyrimidine rings. In both crystalline forms, a hydrated sodium ion bound to the N-7 of a guanine ring was found. Strings of water molecules bridging phosphate anionic oxygen atoms are found along the backbone. The absolute values of the propeller-twist are also different in both structures although the values of the twist are very similar. The results point to the importance of the crystallization conditions when analysing fine structural details like solvation properties of oligomers.

The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control

In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase. Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro. Mutant analysis showed the sequence determinants of this dependence to lie in the region between the −10 box and the transcription start site. As with most promoters subject to stringent control, this portion of the hisR promoter is C–G‐rich. Replacing a C/G bp with T/A at position −7 partially relieves the supercoiling response while changing the sequence between −5 and +1 (‐CCCCCG‐) for ‐GTTAA‐ abolishes the response in vitro and in vivo. The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro. Studies in isoleucine‐starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively. The data presented suggest that the hisR promoters sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e. resistance to melting. We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

In vitro and in vivo antitumour activity and cellular pharmacological properties of new platinum–iminoether complexes with different configuration at the iminoether ligands

In order to widen our knowledge on antitumour trans-[PtCl2(iminoether)2] complexes, we have synthesised two new derivatives, trans-[PtCl2¿E-HN = C(OEt)Me¿2] (1) and trans-[PtCl2¿Z-HN = C(OEt)Me¿2] (2), which differ in the configuration of the iminoether ligands. Isomer 1 showed an in vitro cytotoxicity similar to that of cisplatin in a panel of human tumour cell lines (mean IC50 = 8 and 7.7 microM, respectively), whereas isomer 2 showed a lower activity (IC50 = 14.3 microM). Both 1 and 2 isomers overcame cisplatin resistance of ovarian cancer cell line A2780/Cp8. In agreement with the n-octanol/saline partition ratios, intracellular platinum content (and DNA platination) after a 2-h exposure to equimolar drug concentrations was in the order 1 > 2 >> cisplatin, thus indicating that substitution of imminoethers for ammines determines a major lipophilicity and cellular uptake of the platinum drug. Both 1 and 2 showed a major toxic effect towards an excision repair-defective Drosophila strain, thus indicating cellular DNA as cytotoxic target. Finally, both 1 and 2 were active in vivo against the murine P388 system, but, contrary to the in vitro activity, isomer 2 was slightly more active than 1. On the whole, the results confirm the antitumour activity of trans-[PtCl2(iminoether)2] complexes, and indicate that the configuration of the iminoether ligands may affect the pharmacological properties of this class of complexes.

Conformation of acetylaminofluorene and aminofluorene modified guanosine and guanosine derivatives

Abstract Acetylaminofluorene and aminofluorene modified Guo, GMP, d(GpA) and d(ApG) have been studied by circular dichroism and 1H nuclear magnetic resonance. Aminofluorene modified Guo is preferentially in the anti conformation and acetylaminofluorene modified Guo in the syn conformation. It is proposed that the anti conformation of aminofluorene modified Guo is stabilized by an intra molecular hydrogen bond between the NH group of aminofluorene residue and the 5′-OH group of the sugar. The results on the modified dinucleoside monophosphates are analyzed according to this hypothesis.

Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene

Several studies have shown that the carcinogen N-acetoxy-N-2-acetylaminofluorene (AAAF) reacts in vivo and in vitro with native DNA and that the DNA contains a major (80%) adduct N(deoxyguanosin8-yl)-acetylaminofluorene (dGuo8-AAF) and a minor (20%) adduct 3{deoxyguanosin-N2-yl)-acetylaminofluorene (dGuo-N2-AAF) (reviewed [1,2]). It has been shown that the major alteration induced by the fluorene ring is the creation of locally disorganized regions inside the double helical structure [3-81 . Methods sensitive enough to assay the regions of DNAmodified by a carcinogen at the levels of modification occuring in the ‘in vivo’ carcinogenesis experiments would be of great value. The immunological method has already been shown to be able to detect small modifications in DNA (see e.g. [9,10]). On the other hand, the study of the specificity of the antibodies can bring some knowledge on the conformation of the antigen. For these reasons, we have undertaken a study of the immunogenicity of native DNA after reaction with AAAF. In this paper, we show that native DNA slightly modified by AAAF can induce in rabbits the synthesis of specific antibodies which selectively recognize AAF-substituted DNA. A methods of purification of these antibodies is described. Also, the association constants for the binding of the antibodies and several ligands are reported.

Antiserum to Z-DNA

Nucleic acids duplexes have been thought to be conformationally related to A-DNA or B-DNA. However, X-rays studies on crystals of alternating d(CpG)DNA fragments and on fibers of poly(dG-dC) • poly(dG-dC) have demonstrated a left-handed double helix termed Z-DNA [ 1-4]. In solution, poly(dG-dC) • poly(dG-dC) can adopt the Bor Z-form depending upon the ionic strength of the medium [5-9] . Moreover, it seems possible to join sequences in Band Z-form, respectively [ 10,11 ]. Physicochemical techniques for looking at the Z-form need rather large quantities of material and are hardly useful to in situ studies. On the other hand, immunochemical techniques are very sensitive and there are already many reports on the use of specific antibodies to detect small amounts of nucleosides, modified nucleosides or hybrids in nucleic acids (see [12-181 and references herein). In this paper, we report that one can get rabbit antisera which reacts with poly(dG-dC) • poly(dG-dC) in Z-form and does not react with poly(dG-dC) • poly(dG-dC) in B-form. The rabbits were immunized with a poly(dG-dC) • poly(dG-dC) modified by chlorodiethylenetriamino platinum(II) chloride (dien-Pt). This polynucleotide, in which 12% of the bases are complexed to dien-Pt (on the N(7) of guanine residues), adopts the Z-form in physiological conditions [19] while the Z-form of unmodified poly(dG-dC) • poly(dG-dC) is stable at much higher salt concentration [5].

Z-DNA immunoreactivity in rat tissues

Recently, it has been shown that natural and synthetic deoxynucleotide polymers can adopt a left-handed helical structure (termed Z-DNA) in appropriate conditions (see, for example, refs 1 and 2 and the references therein). In contrast to the more familiar right-handed B-DNA, Z-DNA is strongly immunogenic, and polyclonal and monoclonal antibodies against Z-DNA have been elicited3–6. By using such antibodies, immunoreactivity for Z-DNA has been detected in the polytene chromosomes of two dipteran species7–9, in the macronucleus of a ciliated protozoon10, and in certain plant nuclei (cited in ref. 11). In view of the possible importance of Z-DNA as a genomic regulatory signal7, it would be highly desirable to know whether Z-DNA also occurs in mammals. We have therefore initiated an immunohistochemical study of various rat tissues by using three antisera specific for Z-DNA, and the peroxidase–antiperoxidase technique12 for visualization of tissue-bound antibodies. Here we demonstrate that the nuclei of many, but not all, types of rat cells exhibit Z-DNA immunoreactivity, suggesting that Z-DNA may exist naturally in mammalian chromatin.

In vivo evidence for back and forth oscillations of the transcription elongation complex

We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock. We show that the complex readblocked within an (ATC/TAG)n sequence is in a dynamic equilibrium between upstream‐ and downstream‐ translocated conformers. By increasing the strength of the putative RNA‐DNA hybrid, the ternary complex is readily trapped in the downstream‐translocated conformation, where the melted DNA region is limited to 8 bp. The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5′ end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis‐acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.

Identification of left-handed Z-DNA by indirect immunofluorescence in polytene chromosomes of Chironomus thummi thummi

Abstract In this work, antibodies against Z-DNA were used to stain polytene chromosomes of Chironomus thummi thummi . By indirect immunofluorescence we report the first identification of left-handed conformation of DNA in a band region. The Chironomus pattern also contrasts with the general staining observed in Drosophila . In Chironomus the antibodies to Z-DNA bind to one interband region of the chromosome II and two bands regions of the chromosome IV.