High affinity binding of proteins HMG1 and HMG2 to semicatenated DNA loops
© Gaillard and Strauss; licensee BioMed Central Ltd. 2000
Received: 11 August 2000
Accepted: 9 October 2000
Published: 18 October 2000
Proteins 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.
Here 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.
Of 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.
Proteins HMG1 and HMG2, two of the most abundant non histone proteins, have been known for more than 25 years (for a review see ), and their function has been the subject of varied investigations, especially since it was found that they contain a domain of homology with many proteins implicated in the control of development or of differentiation. As examples of recent studies are their immunocytochemical localization , the deletion of the gene coding for HMG1 by homologous recombination in transgenic mice , the effect of HMG1 or of the HMG domain on the assembly of certain nucleoproteic complexes , the observation of the binding of HMG1 to Oct and Hox proteins [5,6], to nuclear hormone receptors , or to p53 , the influence of HMG1 on the circularization of short DNA fragments [9,10], on V(D)J rearrangement in vitro [11,12], or on transcription [13,14]. HMG1 has also been recently implicated as a mediator of endotoxin lethality . An important way of studying the function and the mechanism of action of proteins HMG1/2 has obviously been the search for molecular partners, and, starting from the assumption that these chromatin proteins probably interact with DNA, many studies have been performed to study the sequences or the DNA conformations with which the proteins interact preferentially. For some HMG-domain proteins, specific binding sequences have been identified , but no such sequences have been found for proteins HMG1 and HMG2 themselves, which interact only weakly with double-stranded DNA. However, it has been shown that HMG1/2 could form complexes with several kinds of non-classical DNA structures: supercoiled circles [17,18,19,20], platinated DNA [21,22], UV-modified DNA , bulge loops [18,24,25], and four-way junctions . As the interactions with four-way junctions were stronger than others, they have been the object of particular studies [18,26,27,28,29,30,31]. Thus, the image of HMG1 that has prevailed is of "an all-purpose DNA-bending, -wrapping, and -looping factor that can be recruited for transcription, DNA repair, and recombination" .
Here we have studied the interactions of HMG1/2 with Form X, and found that these proteins bind much more strongly to semicatenated DNA junctions than to any other known DNA substrate.
As HMG1 and HMG2 were known to interact with many different forms of DNA, we first compared the interactions of HMG1/2 with Form X and with several different DNA structures. Using labelled DNA minicircles as a substrate we observed preferential interactions of certain supercoiled topoisomers with HMG1/2 as described, but these interactions were always much weaker than the interactions with Form X (data not shown). Similarly, the interactions of HMG1/2 with bulge loops made by association of appropriate synthetic oligonucleotides were much weaker than the interactions of HMG1/2 with Form X. In all experiments with minicircles or bulge loops we could only observe a partial retardation of the labelled substrate and E. coli DNA was always an effective competitor of the interactions (not shown). In contrast, under identical conditions, Form X was always entirely retarded, and E. coli DNA was not a competitor.
With our results showing that Form X is the strongest known DNA substrate for HMG1/2 proteins, the general situation with respect to the affinity of HMG1/2 to different DNA conformations can be summarized as follows: HMG1 and HMG2 have an affinity of the order of 106 for double-stranded DNA, bent or unbent, lower than the affinity of most HMG-domain proteins which have affinities high enough to show specific binding sites and complexes by gel retardation. A higher affinity is observed towards bulge loops, supercoiled circles, or platinated DNA. Then, with an affinity of 108 to 109, the four-way junction was the known substrate of highest affinity for HMG1/2, and the suggestions of biological function for these proteins have most often dealt with such interactions. Now we observe a much tighter binding of HMG1 and HMG2 to Form X, with an affinity constant of more than 5 × 1012, and the in vivo significance of interactions observed so far in vitro will have to be studied. The first question is obviously whether structures similar to Form X exist in the cell, at what stages, and at which frequency. Technically, given the stability of Form X and its high affinity for HMG1/2, it should be possible to detect Form X in the cell even if it is rare. It will also be important to determine whether the HMG domain is as important in these interactions as with four-way junctions, and whether other HMG-domain proteins also bind Form X structures.
Of 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, suggesting that the possibility should be studied that such DNA hemicatenanes might exist in the cell nucleus. In addition, a role for the HMG domain in the formation or the stabilization of higher order chromatin structures has often been suggested (reviews in [1,32,38]), and our results go further in the same direction, by suggesting that one of the functions of HMG1/2 might be linked to the remarkable property of Form X to associate two distant regions of the genome in a stable but reversible manner.
Materials and Methods
The formation and purification of Form X has been described in detail elsewhere . Briefly, a DNA fragment containing a tract of poly(CA)· poly(TG) in its center is heat-denatured and allowed to renature in the presence of protein HMG1/2. The complexes formed, in which HMG1/2 is found associated with Form X, are purified by polyacrylamide gel electrophoresis and electroeluted. The protein is then removed by extraction with chloroform in 1 M NaCl and 1% SDS, Form X is ethanol precipitated and redissolved in 0.1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.5.
Four-way junctions were prepared with synthetic oligonucleotides with sequences identical to those used in .
Proteins HMG1 and HMG2
Proteins HMG1 and HMG2 were purified as described . In the first experiments, their concentration was determined by electrophoresis on SDS-polyacrylamide gels, staining with coomassie blue, and comparison with known markers. Later, given the quantitative binding of HMG1 and HMG2 to Form X, a better precision was achieved by quantifying the interactions with known amounts of radioactively labelled Form X.
Interactions were performed in 25 μL of 25 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT plus 100 μg/mL bovine serum albumin to prevent losses of material on tube walls (0.1% Triton X-100 is equally efficient). After addition of 1 μL of HMG1/2 protein, samples were incubated for 30 min. at 37° and loaded on a polyacrylamide gel. HMG and DNA concentrations are indicated in the Figure legends. Unless otherwise noted, electrophoresis was performed in 4% polyacrylamide gels (acrylamide:bis 29:1) in 6.7 mM Tris-acetate, 3.3 mM Na acetate, 1 mM EDTA, pH 7.8 at 4° with buffer recirculation.
We would like to thank Nathalie Delgehyr for her help with experiments with bulge loops, Caroline Perrin for technical assistance, and Susan Elsevier for critical reading of the manuscript. 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.
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