François Strauss

The mitochondrial genome of wild-type yeast cells. V. Genome evolution.

When degraded with the restriction enzymes Hae III or Hpa II, the mitochondrial DNAs from one Saccharomyces carlsbergensis and three genetically unrelated S. cerevisiae wild-type strains yielded 71 to 113 fragments ranging in molecular weight from 10 4 to 4×10 6 . Genome unit sizes, calculated by adding up the molecular weights of all fragments produced by Hae III, Hpa II and, in some cases, Hind II+III and Eco RI, were in the 52 to 54×10 6 range for the three S. cerevisiae strains, whereas a value lower by about 10% was found for the S. carlsbergensis strain. These values are in agreement with the physical size of circular twisted yeast mitochondrial DNA, as determined by electron microscopy (Hollenberg et al. , 1970). Large differences in the electrophoretic patterns of Hae III and Hpa II fragments were found among the DNAs from different S. cerevisiae strains; S. cerevisiae and S. carlsbergensis DNAs showed only very few bands having the same mobility. Such differences appear to originate essentially from additions and deletions in the A+T-rich spacers and to be accompanied by a large preservation of gene order. Unequal crossing-over events at the spacers seem to be the source of additions and deletions and to underlie the evolution of the mitochondrial genome of yeast.

Photographic quantitation of DNA in gel electrophoresis.

Abstract A photographic procedure to quantitate the DNA in bands, obtained by gel electrophoresis after staining with ethidium bromide, is described. The relationship between the film darkening and the intensity of the light hitting the film was determined. The densities, measured in densitometric tracings of the negatives, were converted into fluorescence intensities. The fluorescence was found to be linearly proportional to the amount of DNA. Deviations due to gel overloading, to nonuniform electrophoretic migration and uv illumination, and to photodecomposition of ethidium bromide were investigated.

Sequence-specific single-strand-binding protein for the simian virus 40 early promoter stimulates transcription in vitro.

We have detected, in nuclear extracts of non-infected cultured monkey cells, a protein (protein H16) that binds a specific single-stranded DNA sequence in the early promoter of simian virus 40 (SV40). This protein does not bind double-stranded DNA, nor RNA. In the present paper, the DNA-binding properties of protein H16 and its effects on transcription by RNA polymerase II in vitro have been investigated. The protein binds only to the late strand of the early promoter, within the region of the 21 base-pair repeats, and shows no affinity for any other SV40 sequence. The high percentage of cytosine residues in the late strand in this region appears to be important for recognition by the protein. Protein H16 does not bind the control region of SV40 in negatively supercoiled DNA circles. When bound to the late strand, the protein is displaced from its binding site by reassociation of the early strand with the late strand. Its binding to DNA is not sensitive to methylation of the dinucleotide CG in its binding site. The protein has been purified to near homogeneity by preparative gel retardation, and has an apparent molecular weight of 70,000. Purified protein H16 stimulates transcription by purified RNA polymerase II in vitro. The possible role of sequence-specific single-strand-binding proteins in transcription is discussed.

High affinity binding of proteins HMG1 and HMG2 to semicatenated DNA loops

BackgroundProteins HMG1 and HMG2 are two of the most abundant non histone proteins in the nucleus of mammalian cells, and contain a domain of homology with many proteins implicated in the control of development, such as the sex-determination factor Sry and the Sox family of proteins. In vitro studies of interactions of HMG1/2 with DNA have shown that these proteins can bind to many unusual DNA structures, in particular to four-way junctions, with binding affinities of 107 to 109 M-1.ResultsHere we show that HMG1 and HMG2 bind with a much higher affinity, at least 4 orders of magnitude higher, to a new structure, Form X, which consists of a DNA loop closed at its base by a semicatenated DNA junction, forming a DNA hemicatenane. The binding constant of HMG1 to Form X is higher than 5 × 1012 M-1, and the half-life of the complex is longer than one hour in vitro.ConclusionsOf all DNA structures described so far with which HMG1 and HMG2 interact, we have found that Form X, a DNA loop with a semicatenated DNA junction at its base, is the structure with the highest affinity by more than 4 orders of magnitude. This suggests that, if similar structures exist in the cell nucleus, one of the functions of these proteins might be linked to the remarkable property of DNA hemicatenanes to associate two distant regions of the genome in a stable but reversible manner.

Avoiding adsorption of DNA to polypropylene tubes and denaturation of short DNA fragments.

Two problems can arise when working with small quantities of DNA in polypropylene tubes: first, significant amounts of DNA can become lost by sticking to the tube walls; second, short DNA fragments tend to denature when binding to polypropylene. In addition, DNA also tends to denature upon dehydration. We have found that a simple way to solve these problems is by using polyallomer tubes instead of polypropylene and by avoiding certain salts, such as sodium acetate, when drying DNA.

DNA loops and semicatenated DNA junctions

BackgroundAlternative DNA conformations are of particular interest as potential signals to mark important sites on the genome. The structural variability of CA microsatellites is particularly pronounced; these are repetitive poly(CA) · poly(TG) DNA sequences spread in all eukaryotic genomes as tracts of up to 60 base pairs long. Many in vitro studies have shown that the structure of poly(CA) · poly(TG) can vary markedly from the classical right handed DNA double helix and adopt diverse alternative conformations. Here we have studied the mechanism of formation and the structure of an alternative DNA structure, named Form X, which was observed previously by polyacrylamide gel electrophoresis of DNA fragments containing a tract of the CA microsatellite poly(CA) · poly(TG) but had not yet been characterized.ResultsFormation of Form X was found to occur upon reassociation of the strands of a DNA fragment containing a tract of poly(CA) · poly(TG), in a process strongly stimulated by the nuclear proteins HMG1 and HMG2. By inserting Form X into DNA minicircles, we show that the DNA strands do not run fully side by side but instead form a DNA knot. When present in a closed DNA molecule, Form X becomes resistant to heating to 100°C and to alkaline pH.ConclusionsOur data strongly support a model of Form X consisting in a DNA loop at the base of which the two DNA duplexes cross, with one of the strands of one duplex passing between the strands of the other duplex, and reciprocally, to form a semicatenated DNA junction also called a DNA hemicatenane.

Association of double-stranded DNA fragments into multistranded DNA structures

We have previously observed that double-stranded DNA fragments containing a tract of the tandemly repeated sequence poly(CA). poly(TG) can associate in vitro to form stable complexes of low electrophoretic mobility, which are recognized with high specificity by proteins HMG1 and HMG2. The formation of such complexes has since been observed to depend on interactions of DNA with polypropylene surfaces, with the suggestion that the formation of low mobility complexes might be the result of strand dissociation followed by misaligned reassociation of the repetitive sequences. The data presented here show that at high ionic strength the interactions of DNA with polypropylene are sufficiently strong for DNA to remain bound to the polypropylene surface, which suggests that DNA might also be involved in interactions with hydrophobic molecules in vivo. Under such conditions, low-mobility complexes are found only in the material adsorbed to the polypropylene surface, and all DNA fragments are able to form low-mobility structures, whether or not they contain repetitive sequences. Preventing the separation of strands by ligating hairpin loop oligonucleotides at both ends of the fragments does not prevent the formation of low-mobility complexes. Our results suggest two different pathways for the formation of complexes. In the first, dissociation is followed by misaligned reassociation of repetitive sequences, yielding duplexes with single-stranded end regions that associate to form multimeric complexes. In the second, repetitive as well as nonrepetitive DNA molecules bound to polypropylene adopt a conformation with locally unwound regions, which allows interactions between neighboring duplexes adsorbed on the surface, resulting in the formation of low-mobility complexes.

Structural analysis of hemicatenated DNA loops

BackgroundWe have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA)·poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops.ResultsHere we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation.ConclusionsThe present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA)·poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.

Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains.

EXCISION AND REPLICATION OF MITOCHONDRIAL GENOMES FROM SPONTANEOUS PETITE MUTANTS OF YEAST

ABSTRACT The sequences involved in the excision of mitochondrial genomes of spontaneous petite mutants of yeast from the genomes of wild-type cells have been found to correspond to (CCGG, GGCC) clusters and to sequences in the AT spacers. In addition, results have been obtained on nucleotide sequences which are likely to correspond to the origin of replication of the mitochondrial genome. It is now well established that the mitochondrial genome of spontaneous cytoplasmic petite mutants of Saccharomyces cerevisiae originate from those of parental wild-type cells by a mechanism involving a) the excision of a segment of the latter genomes, and b) its subsequent tandem amplification, as shown in fig. 1; the repeat units of the petite genomes so formed may in turn undergo further deletions leading to secondary petite genomes having shorter repeat units (see Bernardi, 1979, for a brief review). Ten years ago, the excision mechanism was considered to be due to il legitimate, site-specific recombination events in the long AT-spacers (forming 50% of the mitochondrial genome) which were supposed to contain sequence repetitions. The subsequent discovery in the mitochondrial genome of yeast of many short segments extremely rich in GC, the GC clusters, (several of which were likely to be homologous in sequence), raised the possibility that these sequences, later shown to be embedded in the AT spacers, were also involved in the recombination phenomena underlying the excision process. In any case, the basic idea of the model was that the instability of the mitochondrial genome of yeast, (the spontaneous petite mutation has a rate of 1-5% per generation in most strains), was due to the existence in each mitochondrial genome unit of a number of nucleotide sequences having enough homology to allow illegitimate site-specific recombination to take place.