Robert Alazard

Mechanism of toxicity of platinum(II) compounds in repair-deficient strains of Escherichia coli

Abstract The survival of wild-type and repair-deficient Escherichia coli treated with cis-Pt(NH3)2Cl2, trans-Pt(NH3)2Cl2 and [Pt(dien)Cl]Cl (dien = H2NCH2CH2NHCH2CH2NH2) was inversely correlated with the ability of these compounds to inhibit DNA synthesis in different bacterial strains. The relative amounts of these 3 compounds covalently bound to DNA immediately after treatment with the same dose were, respectively, 1:⩾2:1, their relative abilities to inhibit DNA synthesis were 6:1:0 and their relative toxicities toward the wild-type and uvrA strains were 3–5:1:0. More repair synthesis, as measured by density-gradient centrifugation techniques, was observed in wild-type bacteria after treatment with the cis than with the trans isomer whereas no repair synthesis was detected after exposure to [Pt(dien)Cl]Cl. These results are consistent with the hypothesis that cis-Pt(NH3)Cl2 binds to DNA and inhibits DNA synthesis thereby killing the cell. The lower toxicity of this compound toward wild-type bacteria compared with repair-deficient strains is in part a consequence of DNA repair. trans-Pt(NH3)2Cl2 and [Pt(dien)Cl]Cl are less toxic than the cis isomer; this lesser toxicity is not a consequence of low levels of DNA binding or enhanced repair of the lesions but appears to reflect a weaker inhibition of DNA synthesis by these Pt-DNA adducts.

Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator‐binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site‐specific recombinational switches. Furthermore, we show that induction of a Mucte62 prophage in a fis lysogen occurs at a lower temperature than that of a wild‐type strain, and that spontaneous induction of Mucfs62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage λ.

Functional domains of bacteriophage Mu transposase: properties of C-terminal deletions

We have generated a series of 3’deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the λ Pl promoter, were analysed in vivo for their capacity to complement a superinfecting Mu4am phage, both for lytic growth and lysogeny, and for their effect on growth of wild‐type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.

Identification of the ‘NORE’ (N-Oct-3 responsive element), a novel structural motif and composite element

N-Oct-3 is a neuronal transcription factor widely expressed in the developing mammalian central nervous system, and necessary to maintain neural cell differentiation. The key role of N-Oct-3 in the transcriptional regulation of a multiplicity of genes is primarily due to the structural plasticity of its so-called ‘POU’ (acronym of Pit, Oct, Unc) DNA-binding domain. We have recently reported about the unusual dual neuro-specific transcriptional regulation displayed by N-Oct-3 [Blaud,M., Vossen,C., Joseph,G., Alazard,R., Erard,M. and Nieto,L. (2004) J. Mol. Biol., 339, 1049–1058]. To elucidate the underlying molecular mechanisms, we have now made use of molecular modeling, DNA footprinting and electrophoretic mobility shift assay techniques. This combined approach has allowed us to uncover a novel mode of homodimerization adopted by the N-Oct-3 POU domain bound to the neuronal aromatic amino acids de-carboxylase and corticotropin-releasing hormone gene promoters and to demonstrate that this pattern is induced by a structural motif that we have termed ‘NORE’ (N-Oct-3 responsive element), comprising the 14 bp sequence element TNNRTAAATAATRN. In addition, we have been able to explain how the same structural motif can also induce the formation of a heterodimer in association with hepatocyte nuclear factor 3β(/Forkhead box a2). Finally, we discuss the possible role of the NORE motif in relation to neuroendocrine lung tumor formation, and in particular the development of small cell lung cancer.

Post replication repair in an excision defective strain of Escherichia coli following treatment with cis dichlorodiammineplatinum(II)

Abstract The size of the DNA synthetized after treatment of an excision defective E. coli strain with cis -dichlorodiammineplatinum(II) ( cis -PDD) was examinated using sedimentation in alkaline sucrose gradients. DNA synthetized during a 10 minutes pulse after treatment with cis -PDD sediments with a molecular weight lower than control DNA from untreated cells. Post treatment incubation of the cells leads to an increase in the sedimentation rate of this DNA which approaches that of normal DNA. This last process is partially abolished in a uvr B5 rec B21 double mutant. These results suggest that single strand breaks or gaps are produced during treatment and are filled in during further reincubation as part of a post replication repair process.

Study of the expression of UVRA and SSB proteins in vivo in λ hybrid phages containing the uvrA and ssbA genes of Escherichia coli

A 9.3-kb Eco RI fragment obtained by partial digestion of the plasmid pDR2000 and containing the uvrA and ssbA genes was subcloned in the insertion vector lambda gt4. Two hybrid bacteriophages carrying this fragment inserted in opposite orientations were isolated and used to lysogenize a uvrA and an ssbA mutant of Escherichia coli. Both phages conferred to these host bacteria the ultraviolet resistance of the wild-type parent indicating full complementation of the uvrA and of the ssbA defect. Two polypeptides corresponding to the molecular weights of the UVRA protein (115 000 dalton) and of the SSB protein (18 500 dalton) were synthesized and amplified after infection of a UV-irradiated lambda ind- lysogen with these 2 hybrid phages. The UVRA protein was not amplified after infection of a lex A3 host while SSB was still produced in large amount. These results establish that uvrA is repressed by lexA in vivo whereas ssbA is not.

Effects of Platinum Coordination Complexes on Mutants of Escherichia coli Defective in DNA Repair and Genetic Recombination

The effect of three platinum coordination complexes on wild type and repair deficient mutant of Escherichia coli has been investigated. The antitumor drug cis-platinum (II) diamminodichloride (cis-PDD) produces an inhibition of DNA synthesis and reduces the viability of excision repair and recombination defective mutants. The trans isomer (trans-PDD) has less effect than cis-PDD on all of these mutants while [Pt(dien)C1]C1 has no effect. Incorporation of radioactive precursors under non-replicating conditions and density gradient centrifugation analysis of DNA indicate that repair synthesis occurred after treatment of wild type cells with cis-PDD or trans-PDD.