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The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.
HU is one of the major nucleoid-associated proteins involved in bacterial chromosome structure and in all DNA-dependent cellular activities. Similarly to eukaryotic histones, this small dimeric basic protein wraps DNA in a non-sequence specific manner, promoting DNA super-structures. In most bacteria, HU is a homodimeric protein encoded by a single gene. However, in enterobacteria such as Escherichia coli, the presence of two genes coding for two peptidic chains, HUα and HUβ, lead to the coexistence of three forms : two homodimers EcHUα2 and EcHUβ2, as well as a heterodimer EcHUαβ. Genetic and biochemical investigation suggest that each EcHU dimer plays a specific physiological role in bacteria. Their relative abundance depends on the environmental conditions and is driven by an essential, yet unknown, fast outstanding chain-exchange mechanism at physiological temperature. Our goal is to understand this fundamental mechanism from a structural and kinetics standpoint using NMR. For this purpose, the first steps are the assignment of each dimer in their native and intermediate states. Here, we report the backbone assignment of each HU dimers from E. coli at 293 K in their native state.
DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(D) [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P(ramA) of an MDR S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(D) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially.
The MC1 protein is a chromosomal protein likely involved in the DNA compaction of some methanogenic archaea. This small and monomeric protein, structurally unrelated to other DNA binding proteins, bends DNA sharply. By studying the protein binding to various kinds of kinked DNA, we have previously shown that MC1 is able to discriminate between different deformations of the DNA helix. Here we investigate its capacity to recognize particular DNA sequences by using a SELEX procedure. We find that MC1 is able to preferentially bind to a 15 base pair motif [AAAAACACAC(A/C)CCCC]. The structural parameters of this sequence are characterized by molecular dynamics simulation experiments, and the binding mode of the protein to the DNA is studied by footprinting experiments. Our results strongly suggest that the protein realizes an indirect readout of the DNA sequence by binding to the DNA minor groove.
The chromosomal protein MC1 is a monomeric protein of 93 amino acids that is able to bind any DNA but has a slight preferential affinity for some sequences and structures, like cruciform and minicircles. The protein has been irradiated with Ar-36(18+) ions of 95 MeV/nucleon. The LET of these particles in water is close to 270 keV/mu m. We tested the activity of the protein by measuring its ability to form complexes with DNA. We tested the integrity of the protein by measuring the molecular weight of the species formed. Compared with gamma radiation, we observed for the same dose a less efficient inactivation of the protein, a greater protection of the protein by the bound DNA, a lower induction of chain breakage, and a greater production of protein-protein and DNA-protein crosslinks. The results are discussed in terms of the quantitative and the qualitative differences between the two types of radiation : The global radical yield is slightly higher with gamma rays, whereas the density of radicals produced along the particle track is considerably higher with argon ions. (c) 2005 by Radiation Research Society.
The three-dimensional structure of methanogen chromosomal protein 1 (MC 1), a chromosomal protein extracted from the archaebacterium Methanosarcina sp. CHT155, has been solved using H-1 NMR spectroscopy. The small basic protein MC1 contains 93 amino acids (24 basic residues against 12 acidic residues). The main elements of secondary structures are an a helix and five ß strands, arranged as two antiparallel ß sheets (a double one and a triple one) packed in an orthogonal manner forming a barrel. The protein displays a largely hydrophilic surface and a very compact hydrophobic core made up by side chains at the interface of the two ß sheets and the helix side facing the interior of the protein.
The DNA-binding protein MC1 is a chromosomal protein extracted from the archaebacterium. Methanosarcina sp. CHT155. It binds any DNA, and exhibits an enhanced affinity for some short sequences and structures (circles, cruciform DNA). Moreover, the protein bends DNA strongly at the binding site. MC1 was submitted to oxidative stress through gamma-ray irradiation. In our experimental conditions, damage is essentially due to hydroxyl radicals issued from water radiolysis. Upon irradiation, the regular complex between MCl and DNA disappears, while a new complex appears. In the new complex, the protein loses its ability to recognise preferential sequences and DNA circles, and bends DNA less strongly than in the regular one. The new complex disappears and the protein becomes totally inactivated by high doses.
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 ejectrophoresis. 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.
Purpose : To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. Materials and methods : Two structures of the complex were extracted from the PDB databank : crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4’ and H5’) to OH* and of the FSB probabilities, along : (1) DNA in the complex ; (2) DNA in the complex depleted of the repressor ; and (3) a linear DNA having the same sequence. An 80 bp fragment bearing the operator was irradiated alone or in presence of the repressor.
Binding of a protein to its cognate DNA sequence is a key step in the regulation of gene expression. If radiation damage interferes with protein-DNA recognition, the entice regulation profess may be perturbed. We have studied the effect of gamma rays on a model regulatory system, the E. coli lactose repressor-operator complex. We have observed the disruption of the complex upon irradiation in aerated solution. The complex is completely restored by the addition of nonirradiated repressor, hut not by the addition of nonirradiated DNA.
The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1.
The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four-way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA, double helix.
The primary structure of the DNA-binding protein II from Zymomonas mobilis has been determined from data provided by automated Edman degradation of the intact protein and of peptides derived from cleavage at aspartic acid and arginine residues. When compared with the homologous protein isolated from other bacteria, the DNA-binding protein II from Z, mobilis shows many substitutions. Several non-conservative substitutions at positions usually highly conserved in this type of protein probably account for the weaker DNA-binding activity of this protein compared to that of the E coli protein. ((C) Societe francaise de biochimie et biologie moleculaire/Elsevier, Paris).
The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.
Electron microscopy of DNA, either free or complexed with ligands, allows the analysis of local conformational variations along individual molecules. Electron microscopy is unique, in that it has the capacity to determine the average behaviour of a population of molecules observed individually, and can thus provide a better appreciation of variability within the series of molecules than biophysical or biochemical methods. Very encouraging results have been obtained by cryoelectron and near-field microscopies, especially atomic force microscopy, in parallel with traditional techniques for visualizing DNA molecules adsorbed onto a support film. Differences in sample processing procedures and image formation modes render these 3 types of microscopies complementary. The torsional stress of a DNA molecule together with a local curvature induced by the protein MC1 from archaebacteria, can be detected within minicircles comprising 207 base pairs.
Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy, MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.
DNA minicircles of 207 bp were constructed by the ligation of linear restriction fragments in the presence of various concentrations of ethidium bromide. Three topoisomers characterized by linking numbers (Lk) of 20, 19 and 18, and with helical repeats of 10.35, 10.9 and 11.5 bp/turn respectively, were obtained. They are called, respectively, relaxed minicircle or topoisomer 0, topoisomer -1 and topoisomer -2. Owing to the limited flexibility of such small circles, the stress created by the lack of 1 or 2 turns cannot be eliminated by a spatial circle-axis writhing (supercoiling) of the circular molecules. These two undertwisted, stressed topoisomers have to adopt a flat, non-crossed shape, similar to that of the relaxed minicircle. The three minicircles were irradiated with gamma-rays or fast neutrons. The same yields of single-strand breaks, double-strand breaks and alkali-induced single-strand breaks were observed for the three topoisomers showing that their base and sugar moieties are attacked equally by gamma photon- or fast neutron-induced radicals. We conclude that untwisting of a B helix does not modify the radiosensitivity of DNA.
The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.
The binding of Escherichia coli Cyclic AMP Receptor Protein (CRP) to several DNA fragments of about 45 base pairs, bearing the natural lactose or galactose sites, as well as several synthetic related sites, was investigated using fluorescence spectroscopy and gel retardation experiments. The salt dependence of the equilibrium binding constant indicates that CRP makes an identical number of ion pairs with the lac, lacL8 and gal sites although the binding constants are drastically different. However increasing the symmetry of the gal site leads to an increase of the number of ion pairs between the protein and the DNA. A single strand nick was introduced at the centre of a symmetrized gal site and this reduces the binding energy of CRP by about 0.6 Kcal.
We have investigated the effect on the DNA structure of protein MC1, a basic and small polypeptide (M(r) 10700) representing the major chromosomal protein in Methanosarcinaceae. The ability of protein MC1 to strongly favour cyclization upon polymerization of short DNA fragments by T4 DNA ligase indicates that protein MC1 mediates DNA bending. Several negatively supercoiled topoisomers of minicircles were obtained with DNA fragments of 203 and 146 bp, their distribution depends upon the amount of protein MC1 complexed with DNA. In addition, protein MC1 can induce a compaction of a nicked plasmid.
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