Biomedical subjects

J L Sikorav

Publications and source records attributed to J L Sikorav.

At least 19 recordsLinked to original sources

PubMed1999

Cyclization of globular DNA. Implications for DNA-DNA interactions in vivo.

The rate of cyclization of lambda DNA varies over more than 6 orders of magnitude, from 3.2 x 10(-7) s-1 to 2 s-1, in a Tris-EDTA buffer as a function of spermidine concentration. This variation is strictly correlated with the conformation of the chain. The highest rates are obtained when the chain is collapsed into a dense globular state. The effective concentration of the chain ends in the reaction is then 87 000-fold greater than in the random coil state. These results show that DNA globularity must be taken into account to understand biological processes involving intramolecular DNA-DNA interactions.

Bacteriophage lambda↗

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

Precipitation of DNA by polyamines: a polyelectrolyte behavior.

Conditions of double-stranded DNA precipitation by the polyamines spermidine and spermine have been determined experimentally and compared to theoretical predictions. The influence of the concentrations of DNA and added monovalent salt, and of the DNA length has been investigated in a systematic manner. Three regimes of DNA concentrations are observed. We clarify the dependence of these regimes on the monovalent salt concentration and on the DNA length. Our observations make possible a rationalization of the experimental results reported in the literature. A comparison of the precipitation conditions of different kinds of polyelectrolytes suggests a general process. Our experimental data are compared to the "ion-bridging" model based on short-range electrostatic attractions. By starting from the spinodal equation, predicted by this model, and using the limiting form of Manning's fractions of condensed counterions, analytical expressions of the precipitation conditions have been found in the three regimes. Experimental and theoretical results are in good agreement.

Animals↗

PubMed1998

Accelerated cyclization of lambda DNA.

In the presence of spermidine, the DNA molecule of the bacteriophage lambda undergoes a coil-globule transition. We report here that the cyclization of this molecule in its globular state is greatly accelerated (by more than 10(4)-fold) in comparison with the cyclization reaction taking place in the coil conformation.

Bacteriophage lambda↗

PubMed1996

DNA aggregation induced by polyamines and cobalthexamine.

We have studied the precipitation of short DNA molecules by the polycations spermidine, spermine, and cobalthexamine. The addition of these cations to a DNA solution leads first to the precipitation of the DNA; further addition resolubilizes the DNA pellet. The multivalent salt concentration required for resolubilization is essentially independent of the DNA concentration (between 1 microM/ml and 1 mg/ml) and of the monovalent cation concentration present in the DNA solution (up to 100 mM). The DNA aggregates are anisotropic; those obtained in the presence of the polyamines spermidine and spermine generally contain a cholesteric liquid crystalline phase that flows spontaneously. In contrast this phase is never seen in the presence of cobalthexamine. We propose that the ability of polyamines to condense DNA in fluid structures is an essential feature of their biological functions.

Animals↗

PubMed1996

Forces on chromosomal DNA during anaphase.

In the course of anaphase, the chromosomal DNA is submitted to the traction of the spindle. Several physical problems are associated with this action. In particular, the sister chromatids are generally topologically intertwined at the onset of anaphase, and the removal of the intertwinings results from a coupling between the enzymatic action of type II DNA topoisomerases and the force exerted by the spindle. We propose a physical analysis of some of these problems: 1) We compare the maximum force the spindle can produce with the force required to break a DNA molecule, and define the conditions compatible with biological safety during anaphase. 2) We show that the behavior of the sister chromatids in the absence of type II DNA topoisomerases can be described by two distinct models: a chain pullout model accounts for the experimental observations made in the budding yeast, and a model of the mechanical rupture of rubbers accounts for the nondisjunction in standard cases. 3) Using the fluctuation-dissipation theorem, we introduce an effective protein friction associated with the strand-passing activity of type II DNA topoisomerases. We show that this friction can be used to describe the situation in which one chromosome passes entirely through another one. Possible experiments that could test these theoretical analyses are discussed.

Anaphase↗

PubMed1995

Anaphase chromatid motion: involvement of type II DNA topoisomerases.

Sister chromatids are topologically intertwined at the onset of anaphase: their segregation during anaphase is known to require strand-passing activity by type II DNA topoisomerase. We propose that the removal of the intertwinings involves at the same time the traction of the mitotic spindle and the activity of topoisomerases. This implies that the velocity of the chromatids is compatible with the kinetic constraints imposed by the enzymatic reaction. We show that the greatest observed velocities (about 0.1 microns s-1) are close to the theoretical upper bound compatible with both the diffusion rate (calculated here within a probabilistic model) and the measured reaction rate of the enzyme.

Anaphase↗

PubMed1994

A liquid crystalline phase in spermidine-condensed DNA.

Over a large range of salt and spermidine concentrations, short DNA fragments precipitated by spermidine (a polyamine) sediment in a pellet from a dilute isotropic supernatant. We report here that the DNA-condensed phase consists of a cholesteric liquid crystal in equilibrium with a more concentrated phase. These results are discussed according to Flory's theory for the ordering of rigid polymers. The liquid crystal described here corresponds to an ordering in the presence of attractive interactions, in contrast with classical liquid crystalline DNA. Polyamines are often used in vitro to study the functional properties of DNA. We suggest that the existence of a liquid crystalline state in spermidine-condensed DNA is relevant to these studies.

Crystallization↗

PubMed1994

Kinetics of chromosome condensation in the presence of topoisomerases: a phantom chain model.

We discuss the requirement of type II DNA topoisomerase in the process of mitotic chromosome condensation. Using a known model describing the collapse of homopolymers, we propose that the compaction process necessitates a change in the topological state (i.e., a self-knotting) of the chromosomal chain. We argue that the enzymes are necessary to reach the compact metaphase state in a time interval that is much smaller than the time expected in the uncatalyzed process. The folding process is such that the potential entanglement points are localized at particular regions of the chromosome known as the scaffold-associated regions. The concentration of entanglements in the metaphase chromosome is related to the average size of the radial loops. A phantom chain model for the condensation process, in which each potential entanglement point is dealt with by a topoisomerase II molecule, is proposed.

Biophysical Phenomena↗

PubMed1991

Complementary recognition in condensed DNA: accelerated DNA renaturation.

The functional consequences of DNA condensation are investigated. The recognition of complementary strands is profoundly modified by this critical phenomenon. (1) Condensation of denatured DNA greatly accelerates the kinetics of DNA renaturation. We propose a unifying explanation for the effects of several accelerating solvents studied here including polymers, di- and multivalent cations, as well as effects seen with the phenol emulsions and single-stranded nucleic acid binding proteins. Optimal conditions for renaturation at or above the calculated three dimensional diffusion limit are theoretically consistent with a limited search space in the condensed phases. (2) In addition to these effects on association of two single strands, similar condensation acceleration effects can be seen in strand exchange experiments with double stranded DNA without proteins. These may model a mechanism of recombinational protein function.

Bacteriophage lambda↗

PubMed1988

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms.

Amino Acid Sequence↗

PubMed1987

cDNA sequences of Torpedo marmorata acetylcholinesterase: primary structure of the precursor of a catalytic subunit; existence of multiple 5'-untranslated regions.

cDNA clones coding for a catalytic subunit of acetylcholinesterase were isolated from cDNA libraries constructed from Torpedo marmorata electric organ. The nucleotide sequence of the cloned cDNAs codes for a 599-amino acid precursor containing a 24-amino acid signal peptide. This primary structure has been compared with the sequences of Torpedo californica and Drosophila melanogasta acetylcholinesterases, and with that of human butyrylcholinesterase. Genomic blot experiments carried out with cDNA restriction fragments used as hybridization probes are in agreement with the existence of a single gene coding for the different catalytic subunits of Torpedo acetylcholinesterase. Unexpectedly, we observed multiple 5'-untranslated regions, which may contain several initiation codons.

Acetylcholinesterase↗

PubMed1985

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase.

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.

Acetylcholinesterase↗

PubMed1984

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

Acetylcholinesterase↗

PubMed1983

Structural relationships among mouse and human immunoglobulin VH genes in the subgroup III.

The mouse VHIII subgroup is composed of four families which share sequence homology. We isolated a VH germ-line genomic clone, which cross hybridizes with a cDNA probe from one of these families, derived from a myeloma secreting an antigalactan antibody. We report here the nucleotide sequence of the cross hybridizing gene and show that very likely it has an anti-sheep red blood cell specificity. Comparison of its nucleotide sequence with those of the three other VHIII families shows that these genes share segmental homologies of variable lengths. This suggests that interchanges of sequence blocks between VH genes could be an important evolutionary mechanism for diversifying the germ-line repertoire. The strong homology (82%) with human VHIII genes suggests that efficient antibody sequences are strongly conserved. This conservation of homology is particularly striking when compared to the more limited homology (63%) between mouse and human C kappa genes.

Animals↗

PubMed1981

Mouse heavy chain variable regions: nucleotide sequence of a germ-line VH gene segment.

We have constructed a library of Balb/c mouse embryo DNA in the vector Charon 4A. The library was searched for sequences homologous to the VH region of a cloned cDNA of the UPC10 heavy chain mRNA. In this paper, we describe the structure and the partial nucleotide sequence of one of such clones (VH441). The nucleotide sequence of this germ-line gene indicates that it encodes amino-acids 1-98 of the X44 and J601 galactan-binding VH regions, but that it differs from the UPC10 VH segment by four single base changes. The VH gene appears to contain a 101 bases long intervening sequence within a precursor sequence identical to the precursor sequence of UPC10. The 3' non coding sequence of the V gene contains the two conserved sequences found in embryonic V DNA segments, CACAGTG and ACATGAACC, separated by 23 nucleotides and a sequence CACTGTG separated by 33 nucleotides from the first heptamer.

Animals↗

PubMed1981

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs.

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.

Animals↗