DNA loops and semicatenated DNA junctions

Abstract

Alternative 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. Formation 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. Our 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.

Background

Alternative 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.

Results

Formation 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.

Conclusions

Our 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.

Background

] were drawn to our attention for two reasons. First, they migrated near the regular double-stranded form of the fragment, suggesting that they might correspond to double-stranded, not multi-stranded, structures. Second, they were bound with high affinity by proteins HMG1 and HMG2, two abundant non-histone nuclear proteins for which no double-stranded DNA substrate with such a high affinity was known. Here we describe a mechanism of formation of these structures, and their characterization as DNA loops at the base of which the DNA duplexes form a unique knot in which one of the strands of one duplex passes between the strands of the other duplex, and reciprocally, to form a semicatenated DNA junction, also called a DNA hemicatenane.

Results

] were drawn to our attention for two reasons. First, they migrated near the regular double-stranded form of the fragment, suggesting that they might correspond to double-stranded, not multi-stranded, structures. Second, they were bound with high affinity by proteins HMG1 and HMG2, two abundant non-histone nuclear proteins for which no double-stranded DNA substrate with such a high affinity was known.

. A detailed analysis of the relative affinities of HMG1/2 to different DNA substrates (double-stranded or single-stranded DNA, loops, minicircles, cruciform, Form X) will be presented elsewhere (C.G. and F.S., in preparation).

, lane 2). In the absence of HMG1/2, this same process gives no Form X or hardly detectable amounts of Form X.

]. Starting with this hypothesis, in an attempt to measure the length of such loops, we set out to determine the linking number of DNA in Form X, i.e. the number of times one strand turns around the other strand in such a structure. Indeed, if Form X contains unpaired regions, the linking number is expected to decrease by one unit for each unpaired turn of double helix (10.5 bp).

), circular Form X is extremely stable and is not modified by incubation at 100°C (nor by alkaline pH, result not shown). Neither is it modified by incubation with calf thymus topoisomerase I or by human topoisomerase II, suggesting that it contains no superhelical stress. Since circular Form X does not correspond to any band in the series of topoisomer markers, we then considered that Form X might contain a large number of negative supercoils, larger than in the most supercoiled of the marker topoisomers, and that the corresponding torsional stress was absorbed and constrained by a change of conformation strictly limited to the poly (CA) · poly (TG) region and stabilized by supercoiling of the small circles. For example, a change of conformation from B-DNA to Z-DNA might have corresponded to this hypothesis.

, lanes 3 and 4). Therefore the hypothesis of a global change of conformation of poly(CA) · poly(TG) induced or stabilized by supercoiling had to be ruled out. This experiment also shows with no ambiguity that Form X contains two ends only, since ligation of Form X with the vector yields almost exclusively monomeric circles, even in the presence of an excess of vector. Therefore Form X can only be a two-stranded structure.

shows that Form X remains stable in molecules with closed ends, and resists heating to 100°C and treatment by alkaline pH (lanes 4 and 5), as do circular molecules, but unlike open linear molecules. It should also be noted that upon adding a hairpin oligonucleotide at one end only, Form X is not as stable and can be dissociated by heat treatment (lane 7), although not as easily as when it is contained in an open linear fragment.

. Several parameters of such a structure can vary, including the location of the junction within the repetitive nucleotide sequence, the size of the loop, and the DNA linking number inside the loop. This is probably the explanation for the number of different bands shown by Form X on a polyacrylamide gel, where up to seven bands can be seen depending on the gel concentration.

] which should facilitate the formation of a loop in the central region. On the other hand, the known affinity of HMG1/2 for DNA junctions should help stabilize the transient junction before complete pairing of the non-repetitive terminal regions.

]. The possibility for producing such structures in vitro and their remarkable stability should now allow the study of their characteristics, which should, in turn, facilitate the investigation of their significance in vivo. It should be noted that the repetitive sequence is required only because of the particular process used to prepare Form X, but that there should be no theoretical objection to the existence of such structures with non-repetitive sequences.

]), and the semicatenated loop described here is certainly among the most stable of all DNA loops observed so far.

Formation of Form X by strand reassociation in the presence of HMG1/2. The DNA fragment, labelled on its TG strand, was heat-denatured and allowed to reassociate in the presence of protein HMG1. By electrophoresis on a polyacrylamide gel, complexes between Form X and HMG1 are obtained (lane 1), and can be dissociated by SDS to yield free Form X (lane 2).

], yielding a series of topoisomers containing increasing numbers of negative supercoils (lanes 1-4), with up to 5 negative superturns for the most supercoiled topoisomer. In lanes 8 and 9, circularized Form X and topoisomers 0 and -1 were analyzed after incubation for 5 min. at 100°C. Note that circularized Form X does not migrate like any of the topoisomer markers.

Analysis of the products on a 4% polyacrylamide gel, to show that Form X has remained stable after insertion in plasmid pE10. Lanes 1 and 2: pE10 containing Form X or the regular fragment, respectively. Lane 3: pE10 containing Form X was redigested with EcoRI + ClaI, the 120 bp fragment is recovered as Form X. Lane 4: pE10 containing Form X was incubated 2 min at 100°C and redigested with EcoRI + ClaI, Form X is recovered, showing that it is resistant to 100°C when inserted in a circular molecule. Lanes 5 and 6: redigestion of pE10 containing the regular linear form of the 120 bp fragment, without or with previous incubation at 100°C, respectively. The regular form of the 120 bp fragment is recovered, as expected. Lanes 7 and 8: controls showing respectively Form X and the regular 120 bp linear fragment used in these experiments.

Form X on a linear fragment with closed ends. Hairpin oligonucleotides were added by ligation to the ends of purified Form X. The ligation products were gel-purified and analyzed on a polyacrylamide gel with or without preincubation at 100°C or in 0.1N NaOH. Lanes 1 and 2: linear fragment with closed ends, unincubated (lane 1) or incubated at 100°C (lane 2). Lanes 3-5: Form X with closed ends, unincubated (lane 3), incubated at 100°C (lane 4), or incubated in 0.1N NaOH (lane 5). Lanes 6 and 7: Form X with one end closed and the other end open, unincubated (lane 6) or incubated at 100°C (lane 7). It is observed that Form X with both ends closed is completely resistant to denaturation (lanes 4 and 5).

Model of Form X: a DNA loop with a semicatenated DNA junction. After denaturation, the reassociation of the strands of a DNA fragment containing the sequence poly (CA) · poly (TG) can occur with a shift in the repetitive sequence. In such a case, the reassociation is expected to pause when it reaches the sides of the repetitive region, allowing one of the single-stranded ends to insert into the fork formed by the two single strands at the opposite end. This process is facilitated by HMG1 or HMG2, and possibly also by the presence of complementary sequences left on both sides of the double-stranded region. The final result is a loop at the base of which two DNA duplexes cross, forming a knot in which one of the strands of one duplex passes between the two strands of the other duplex, and reciprocally, to form a semicatenated DNA junction.

Conclusions

). Structures containing DNA hemicatenanes had been previously suggested to exist in the cell but had not been isolated before. The possibility to prepare such structures, combined with their remarkable stability, should allow one to study their evolution and their possible function when introduced into living cells.

Form X: a DNA loop with a semicatenated DNA junction.

DNA fragments

) and were prepared by standard techniques. To close the ends of linear DNA fragments, 26 nucleotide-long synthetic hairpin oligonucleotides with appropriate ends were used.

Form X

P end labelled DNA fragment, in 5 μL of 10 mM Tris-HCl, 1 mM EDTA, pH 7.5, was denatured at 100°C for 2 min, added as quickly as possible to 20 μL of a solution containing the reassociation buffer and ~ 10 ng of HMG protein, and allowed to renature for 45 min. at 37°C. The conditions of reassociation were: 50 mM NaCl, 25 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM EDTA, 100 μg/mL bovine serum albumin. This reassociation process yields complexes between Form X and HMG1/2, which were purified by electrophoresis in 4% polyacrylamide gels (acrylamide:bis 30:1) in 6.7 mM Tris-acetate, 3.3 mM Na acetate, 1 mM EDTA, at 4°C with buffer recirculation. The complexes were electroeluted, proteins removed by chloroform treatment in 1% SDS and 1 M NaCl, and Form X ethanol precipitated and redissolved in 10 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, pH 7.5. The presence of 0.1 M NaCl was found to stabilize Form X, presumably by stabilization of base pairing in the terminal double-stranded regions.

Acknowledgements

We would like to thank Luigi Jonk, Nathalie Delgehyr, and Sandrine Jaouen for their help at various stages of this work. We are grateful to Susan Elsevier for critical reading of the manuscript. C.G. would also like to thank Prof. Alexander Rich (M.I.T.), in whose laboratory bands X were first observed. This work was made possible in part by grants from the Association Française contre les Myopathies, the Ligue Nationale Française Contre le Cancer, and the Association pour la Recherche contre le Cancer.