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Y Timsit

Publications and source records attributed to Y Timsit.

12 recordsLinked to original sources
PubMed1998

DNA crossovers and type II DNA topoisomerases: A thermodynamical study.

We present a theoretical study of the interaction of tight DNA crossovers with eukaryotic type II DNA topoisomerases. A quantitative analysis of the role of the enzyme during anaphase first shows that a tight DNA crossover should be an intermediate of the strand-passage reaction. We then focus on the initial steps of the strand-passage reaction in vitro which lead to the formation of a ternary complex ES1S2 between the enzyme and a tight DNA crossover (where E is the enzyme, S1 (respectively S2) is the first (respectively the second) DNA segment bound by the enzyme, and S1S2 is a tight crossover). This formation can be described by three equilibrium association constants: KS1 (for the reaction E+S1left arrow over right arrow ES1), KS2 (for ES1+S2left arrow over right arrow ES1S2), and KS (for E+S1S2left arrow over right arrow ES1S2) Using published experimental data obtained on the Drosophila enzyme, we derive rough estimates for the intrinsic equilibrium constants KS1 ( approximately 2.5x10(6) M-1) and KS2 ( approximately 10(4) M-1) and for Ks. The huge value found for Ks, about 5x10(16) M-1, suggests that the ternary complex bears a close resemblance with a transition state complex, and is consistent with the notion of a capture of the crossover by a protein clamp. We give a theoretical description of analogues of tight DNA crossovers which consist of two DNA segments stabilized by a covalent crosslinking. Such analogues are predicted to bind the enzyme with a high affinity and should be useful tools for the study of the enzyme.

Anaphase↗
PubMed1998

Symmetry and chirality in topoisomerase II-DNA crossover recognition.

Several experimental data support the notion that the recognition of DNA crossovers play an important role in the multiple functions of topoisomerase II. Here, a theoretical analysis of the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers is performed, using the crystal coordinates of the docking partners. The DNA crossovers are assumed to be clamped into the central hole of the enzyme. Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein-DNA interactions, this analysis shows that two geometric solutions could exist, depending on the chirality of the DNA crossovers. In the first one, the two DNA segments are symmetrically recognized by the enzyme while each single double helix binds asymmetrically the protein dimer. In the second one, each double helix is symmetrically recognized by the protein around its dyad axis, while the two DNA segments have their own binding modes. The finding of potential DNA-binding domains which could interact with the crossovers provides structural supports for each model. The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA-binding site of histone H5 which binds DNA crossover is of particular interest. Each solution, which is consistent with different sets of experimental data found in the literature, could either correspond to different functions of the enzyme or different steps of the reaction. This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes, and raises new questions about the relationships between the mechanism of strand-passage and strand-exchange catalyzed by the enzyme.

Binding Sites↗
PubMed1998

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.

5-Methylcytosine↗
PubMed1998

Non-monooxygenase cytochromes P450 as potential human autoantigens in anticonvulsant hypersensitivity reactions.

Antibodies recognizing rat cytochrome P450 (CYP) 3A1 but not the closely related human CYPs 3A4/5 have been identified in the sera of patients with hypersensitivity reactions to phenytoin and carbamazepine. Comparison of the mapped epitope to the comparable region in CYP3A4 revealed that Leu361 was essential for antibody recognition because of L361V mutation (mimicking human EYLDMVVNETLRL) abolished immunoreactivity. To identify alternative human autoantigens, a site-directed mutagenesis strategy was employed to identify amino acids critical for antibody recognition. A protein database search with the consensus sequence, DxVLxETLxx, from immunoblot analysis produced CYP8 (prostacyclin synthase), CYP5A1 (thromboxane synthase), CYP27 and CYP7A1 (cholesterol 7 alpha-hydroxylase) as possible candidates; considerable homology was also observed with the fungal CYP52A subfamily. Immunoblotting with patient sera and fragments of each candidate autoantigen expressed as Escherichia coli gene 10 fusion proteins confirmed CYP8 and CYP5A1 as possible antigens, and revealed the presence of IgG1 and IgG3 antibodies against a construct mimicking fungal CYP52A10. All patient sera contained IgG4 antibodies against CYP8, CYP5A1 and the fungal mimic suggestive of continual antigenic challenge. In genetically susceptible individuals, prior infectious challenge may be a determinant of risk for the development of anticonvulsant hypersensitivity reactions and has been incorporated into a model investigating the pathogenesis of these events.

Amino Acid Sequence↗
PubMed1997

Effect of cytosine methylation on DNA-DNA recognition at CpG steps.

Although DNA methylation is a fundamental mechanism for repressing genetic activity, the influence of methyl groups on DNA conformation is found to be small. In this study, the role of cytosine methylation is analysed in the context of DNA condensation by examining its influence on DNA-DNA recognition processes. Previously CpG sites were found to act as sequence determinants for the close and specific self-fit of B-DNA helices into cross-overs. In the present study, the crystal structure of the B-DNA dodecamer d(ACCGCCGGCGCC) methylated at its central CpG sequence shows that the methyl groups do not interfere with DNA self-fitting. In contrast, the two methyl groups form a clamp, which traps the incoming phosphate in the groove-backbone interaction. This geometry allows the formation of two new C-H...O hydrogen bonds between the methyl groups and the anionic oxygen atoms of the phosphate, which may further stabilize the interaction. This finding relates cytosine methylation to the formation of higher-order DNA structures and could provide new insights for understanding the mode of action of DNA methylation in genetic inactivation.

Crystallography, X-Ray↗
PubMed1995

Self-fitting and self-modifying properties of the B-DNA molecule.

X-ray structure analysis of oligonucleotides shows that self-fitting of B-DNA molecules by groove-backbone interaction can trigger modification of the secondary structure of the double helix in a sequence-dependent manner, leading to a "pre-melted" transition state. This work reveals that some sequences respond to the DNA-DNA intermolecular interactions by forming rearranged H-bonding schemes which stabilize the transition states. This study suggests that the close and specific approach of DNA segments occurring in genome packaging, DNA looping, synapsis formation or supercoiling can contribute directly to the secondary structure changes needed for DNA processing.

Base Composition↗
PubMed1994

DNA self-fitting: the double helix directs the geometry of its supramolecular assembly.

Groove-backbone interaction is a natural and biologically relevant mechanism for the specific assembly of B-DNA double helices. Crystal engineering and crystal packing analysis of oligonucleotides of different sizes and sequences reveal that the sequence-dependent self-fitting of B-DNA helices is a dominant constraint for their ordered assembly. It can override the other intermolecular interactions and impose the overall geometry of the packing. Analysis of experimental examples of architectural motifs formed by the geometric combination of self-fitted DNA segments leads to general rules for DNA assembly. Like a directing piece for a supramolecular 'construction set', the double helix imposes a limited number of geometric solutions. These basic architectural constraints could direct, in a codified manner, the formation of higher-order structures. DNA architectural motifs exhibit new structural and electrostatic properties which could have some implications for their molecular recognition by proteins acting on DNA.

Base Sequence↗
PubMed1991

Base-pairing shift in the major groove of (CA)n tracts by B-DNA crystal structures.

the crystal packing of the B-DNA dodecamer d(ACCG-GCGCCACA).d(TGTGGCGCCGGT) is characterized by the reciprocal fit of double helices with specific base-backbone interactions in the major groove. Cooling the crystals below -10 degrees C stabilizes a new conformational state with a long-range sequence-dependent one-step shift in the major-groove base pairing. The tilt of the bases leads to the disruption of the Watson-Crick pairing in the major groove and to the formation of interactions with the 5' neighbour of their complement. This alteration propagates along the helical axis over more than half a turn. As a result, the molecular structure is normal when seen from the minor groove side and mismatched in the major groove. Comparison with a parent isomorphous dodecamer structure corresponding to the codon 10-13 of the c-Ha-ras proto-oncogene show that this new structural feature is sequence dependent and clearly favoured by (CA)n tracts. As(CA)n tracts of DNA are involved both in recombination and in transcription, this new recognition pattern should be considered in the analysis of the various processes involving the reading of the genetic information.

Base Sequence↗
PubMed1991

Groove-backbone interaction in B-DNA. Implication for DNA condensation and recombination.

DNA self-fitting is revealed by the study of intermolecular contacts found in the crystal packing of a dodecamer where the helices are locked together by a reciprocal groove-backbone interaction and form a crossed structure. It is proposed that it could be a model for DNA-DNA interaction in several biological processes such as the node of supercoiled DNA and synapsis in recombination. The main topological and symmetrical features of this crossed structure are described and the symmetry-homology relationships are analyzed in the more general case of B-DNA interacting helices. Model-building of Holliday junctions with minimal change from the starting crystal coordinates of the crossed structure leads to at least three different solutions. These various models are compared from the point of view of their symmetry and topology, in the light of their branch migration and resolution properties. In addition, a model for a self-favored reciprocal unwinding mechanism based on the experimentally observed structural alterations, such as the packing-induced opening of G.C base-pairs is proposed. In this model, the phosphate groups of the invading backbone trigger the opening of the base-pairs of the other helix, by pulling cytosine or adenine bases out of the major groove after binding to their amino group.

Base Sequence↗
PubMed1989

Unusual helical packing in crystals of DNA bearing a mutation hot spot.

The target sequence of the restriction enzyme NarI (GGCGCC) is a hot spot for the -2 frameshift mutagenesis (GGCGCC----GGCC) induced by the chemical carcinogens such as N-2-acetyl-aminofluorene. Of the guanine residues, all of which show equal reactivity towards the carcinogen, only binding to the 3'-most proximal guanine within the NarI site is able to trigger the frameshift event. We selected the non-palindromic dodecamer d(ACCGGCGCCACA), whose sequence corresponds to the most mutagenic NarI site in pBR322 DNA; for X-ray structure analysis. Its molecular structure determined at 2.8 A resolution reveals significant deviations from the structure of canonical B-form DNA, with partial opening of three G-C base pairs, high propeller twist values and sequence-dependent three-centred hydrogen bonds. This crystal structure shows a novel kind of packing in which helices are locked together by groove-backbone interactions. The partial opening of G-C base pairs is induced by interactions of phosphate anionic oxygen atoms with the amino group of cytosine bases. This provides a model for close approach of DNA molecules during biological processes, such as recombination.

Base Sequence↗