Biomedical subjects

M Grigoriev

Publications and source records attributed to M Grigoriev.

14 recordsLinked to original sources

PubMed2004

Single-molecule study of RuvAB-mediated Holliday-junction migration.

Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximately 6-kb DNA molecules of identical sequence, and we measured the migration rate at 37 degrees C and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximately 43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).

Bacterial Proteins↗

PubMed2000

Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Acetyltransferases↗

PubMed2000

Crystallographic, X-ray absorption, and IR studies of solid- and solution-state structures of tris(nitrato) N,N,N',N'-tetraethylmalonamide complexes of lanthanides. Comparison with the Americium complex.

To fine-tune the design of optimized donor ligands for nuclear waste actinide selective extraction, both electronic and molecular structures of the actinide complexes that are formed must be investigated. In particular, to achieve the selective complexation of transplutonium 3+ ions versus lanthanide 3+ ions is one of the major challenges, given the chemical similarities between these two f-element families. In this work, the structure of solvent-phase M(NO3)3(TEMA)2 complexes (Ln = Nd, Eu, Ho, Yb, Lu, Am; TEMA = N,N,N',N'-tetraethylmalonamide) was investigated by liquid-phase spectroscopic methods among which extended X-ray absorption fine structure played a major role. In addition, the crystal structures of the species Nd(NO3)3(TEMA)2 and Yb(NO3)3(TEMA)2 have been determined by X-ray diffraction. Nd(NO3)3(C11N2O2H22)2 crystallizes in the monoclinic system (P2(1) space group; a = 11.2627(4) A, b = 20.5992(8) A, c = 22.2126(8) A; alpha = gamma = 90 degrees, beta = 102.572(1) degrees; Z = 6), and Yb(NO3)3(C11N2O2H22)2 crystallizes in the orthorhombic system (P2(1)2(1)2(1) space group; a = 9.3542(1) A, b = 18.1148(2) A, c = 19.7675(2) A; alpha = beta = gamma = 90 degrees; Z = 4). In the solvent phase, the metal polyhedron was found to be similar to that of the solid-state complex Nd(NO3)3(TEMA)2 for M = Nd to Ho. For M = Yb and Lu, a significant elongation of one nitrate oxygen bond was observed. Comparison with measurements on the Am(NO3)3(TEMA)2 complex in ethanol has shown the similarities between the Nd3+ and Am3+ coordination spheres.

Journal Article↗

PubMed2000

A cruciform structural transition provides a molecular switch for chromosome structure and dynamics.

The interaction between specific sites along a DNA molecule is often crucial for the regulation of genetic processes. However, mechanisms regulating the interaction of specific sites are unknown. We have used atomic force microscopy to demonstrate that the structural transition between cruciform conformations can act as a molecular switch to facilitate or prevent communication between distant regions in DNA. Cruciform structures exist in vivo and they are critically involved in the initiation of replication and the regulation of gene expression in different organisms. Therefore, structural transitions of the cruciform may play a key role in these processes.

Base Pairing↗

PubMed1998

Migration of a Holliday junction through a nucleosome directed by the E. coli RuvAB motor protein.

Chromatin plays a critical role in regulating access to DNA by proteins that direct recombination and repair. The E. coli RuvAB protein complex promotes branch migration of the Holliday junction recombination intermediate. The ability of RuvAB to negotiate passage of the junction through nucleosomal DNA is examined. The model system involves the formation of a Holliday junction positioned upstream of a nucleosome. Unassisted, the junction is blocked by a histone octamer. In the presence of RuvAB and ATP, rapid branch migration through the nucleosome is observed. It results in disruption of the histone-DNA interactions leading to the removal of the octamer from the junction intermediate. These results suggest that eukaryotic DNA motor proteins analogous to RuvAB could function during recombination to promote branch migration through chromatin.

Bacterial Proteins↗

PubMed1997

A histone octamer blocks branch migration of a Holliday junction.

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.

Animals↗

PubMed1996

Peptide nucleic acids directed to the promoter of the alpha-chain of the interleukin-2 receptor.

Two 10-mer oligopyrimidine peptide nucleic acids (PNAs) were designed to interfere with IL-2R alpha promoter expression by binding to the regulatory sequences overlapping SRF and NF-kappa B transcription factor sites. Specific complexes were formed on each target sequence, and clearly involved (1) Hoogsteen hydrogen bonds as shown by experiments in which the purine strand of a single or double-stranded target was substituted with 7-deazadeoxyguanosine, (2) P-loop formation on double-helical DNA as evidenced by susceptibility to a single-strand-specific nuclease. When formed on a single-stranded DNA target, these highly stable complexes were responsible for efficient physical blockage of T7 DNA polymerase elongation on the template DNA containing the target oligopurine sequence. On a double-stranded target, these complexes only formed at low ionic strength and were slowly dissociated at physiological ionic strength (pH 6.5) with a t1/2 of 6.5-7 h. The salt-dependent instability of preformed complexes on a plasmid target was probably the critical factor responsible for their lack of significant sequence-specific effect on IL-2R alpha promoter activity inside living cells.

Binding Sites↗

PubMed1996

Detection of covalent triplex within human cells.

Triple helix-forming oligonucleotides covalently linked to psoralen can be specifically cross-linked to both strands of DNA at the triplex-duplex junction following UV irradiation. We have previously shown that a 15mer psoralen-oligonucleotide conjugate forming a triple helix on the promoter of the alpha subunit gene of the interleukin-2 receptor inhibits transcription of reporter plasmids transfected into living cells after irradiation. In the present work, we directly demonstrate covalent triple helix formation at the target site inside cells. A primer extension assay using Taq polymerase was developed to quantitate the DNA which had reacted with the psoralen of the triple helix-forming oligonucleotide. Photoaddition of the psoralen at the DNA target site was demonstrated, not only when the preformed triplex was electroporated inside cells, but also when the oligonucleotide was added to the culture medium after plasmid electroporation and before irradiation of the cells.

Base Sequence↗

PubMed1995

Oligonucleotide-directed switching of DNA polymerases to a dead-end track.

During DNA replication, the presence of oligonucleotides with partial homology to the template strand was shown to induce a switch of the polymerase from the normal template to the oligonucleotide. The latter acted as a dead-end template and led to abortive replication. The only prerequisite was that the oligonucleotide could form 7-9 base pairs with the newly synthesized DNA strand in order to switch templates. The switch occurred when base pairing of the oligonucleotide could take place with the 3'-end of the newly synthesized strand. These results show that oligonucleotides used in antisense or antigene strategies could have unexpected effects on replication. In addition, oligonucleotide-directed abortive replication might play an inhibitory role during PCR experiments on long DNA templates and lead to the amplification of truncated fragments.

Base Sequence↗

PubMed1993

The serum unresponsive Rous sarcoma virus promoter sustains a high serum response factor-dependent transcription in vitro.

CArG boxes are cis-regulatory elements which are represented both in serum responsive and unresponsive promoters. Here we show that the RSV Long Terminal Repeat contains two CArG boxes, which were efficiently recognised by purified Serum Response Factor, although they remained unresponsive to serum in transient transfection assays. However, RSV CArG boxes were as efficient as c-fos Serum Response Element in mediating a Serum Response Factor-dependent transcription in vitro. Thus, the fact that a CArG box is able to bind Serum Response Factor in an active form is insufficient for serum responsiveness in vivo.

3T3 Cells↗

PubMed1993

Repression of c-fos promoter by MyoD on muscle cell differentiation.

Terminal differentiation and cell proliferation are in many cases, as in muscle cells, mutually exclusive processes. While differentiating myoblasts are withdrawn from the cell cycle, myogenesis is inhibited by some mitogens and overexpression of some oncogenes, including proto-oncogene c-fos (which expresses a growth-associated protein constituting the regulatory factor AP-1 in conjunction with c-Jun). MyoD, a muscle-specific transcription factor of the basic helix-loop-helix family, acts at both levels because it triggers a muscle differentiation programme in non-muscle cells, and induces a complete block of cell proliferation. Antagonistic interaction between MyoD and c-Jun has been demonstrated. We here show that c-fos expression greatly decreases upon muscle cell differentiation, concomitant with MyoD-induced activity. We have identified a MyoD-binding site overlapping with the serum-responsive element in the c-fos promoter. We demonstrate that MyoD can act as a negative regulator for c-fos transcription by blocking serum responsiveness through this binding site. These data suggest that the MyoD negative effect on cell growth could be partly mediated by transcriptional inactivation of growth-responsive genes.

3T3 Cells↗

PubMed1993

Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites.

Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner. We have recently designed psoralen-oligonucleotide conjugates, which, upon binding to double-stranded DNA sequences via triple helix formation, may be cross-linked in vitro to both strands of the DNA following UV irradiation. A psoralen-oligonucleotide conjugate was targeted to the promoter of the alpha subunit of the interleukin 2 receptor (IL-2R alpha) gene. The triple helix site overlaps the binding site for the transcription factor NF-kappa B, which activates transcription from the IL-2R alpha promoter. After UV irradiation, the oligonucleotide conjugate becomes cross-linked to the target site and inhibits transcription of reporter plasmids transfected in live cells. Inhibition is observed when UV-induced cross-linking occurs both in vitro (before transfection) and in vivo (after transfection). We directly demonstrate that this inhibitory effect is due to triple helix formation at the target site, since a mutant of the promoter, to which oligonucleotide binding was inhibited, was not affected by the psoralen-oligonucleotide conjugate after UV irradiation. In addition, we demonstrate that site-specific cross-linking upstream of the promoter has no effect on transcription.

Base Sequence↗

PubMed1993

Inhibition of interleukin-2 receptor alpha-subunit gene expression by oligonucleotide-directed triple helix formation.

Oligonucleotide-directed triple helix formation represents a promising approach to block gene expression at the transcriptional level. We have previously shown [10] that a triple-helix-forming oligonucleotide was able to inhibit promoter function of reporter constructs in live cells, provided that the oligonucleotide was covalently linked to an intercalating agent which stabilizes triple-helical complexes. In order to demonstrate that this inhibitory effect was due to triple helix formation, we have mutated the oligonucleotide target site in the promoter of the interleukin-2 receptor alpha-chain gene. The mutated version of the promoter does not bind, and is not inhibited by the oligonucleotide, demonstrating that the observed inhibition of the wild-type promoter is indeed due to triple helix formation within cells.

Gene Expression↗

PubMed1992

A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.

Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.homopurine sequence located in the upstream regulatory region of the gene encoding the interleukin-2 receptor alpha chain (p55, IL-2 R alpha). Methylation of the cytosines strongly stabilized the triplex. Further attachment of an intercalating agent (acridine) dramatically increased the stability of the triplex, as assessed by Tm measurements or by band shift assays. Furthermore, the acridine-derivatized oligonucleotide was more efficient in competing away high affinity DNA-binding proteins, as assessed by restriction enzyme inhibition assays. Using a novel footprinting assay, we have further shown that the interaction of the methylcytosine-substituted, acridine-derivatized oligonucleotide with a plasmidic target, harboring the IL-2 R alpha regulatory region, remains highly sequence specific, occurs at physiological pH and is independent of the superhelicity of the plasmid. Acridine derivatization did not impair the exquisite target specificity of triplex formation, since the derivatized oligonucleotide inhibited the binding of nuclear proteins to the overlapping NF kappa B enhancer sequence on an IL-2 R alpha target and not on the related human immunodeficiency virus long terminal repeat target. Finally, the oligonucleotide inhibited the NF kappa B-dependent tax-induced transcriptional activation of the IL-2 R alpha chloramphenicol acetyltransferase construct in live cells, whereas it did not have any effect on a human immunodeficiency virus long terminal repeat chloramphenicol acetyltransferase construct. We conclude that this modified oligonucleotide acts as a transcriptional repressor for the IL-2 R alpha gene via triple helix formation with regulatory sequences.

Acridines↗