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Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach.

Rivera-Mulia JC, Aranda-Anzaldo A - DNA Res. (2010)

Bottom Line: Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence.The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced.We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, Toluca, Edo. Méx., México.

ABSTRACT
Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

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Properties of naked DNA loops attached to the NM. (A) Drawing illustrating the local topology along a typical supercoiled DNA loop that correlates with both distance relative to the NM and sensitivity to DNase I. (B) Phase contrast micrograph showing the NM of a hepatocyte nucleoid. (C) Fluorescence micrograph showing the DNA halo around the NM of a hepatocyte nucleoid caused by the unwinding of the supercoiled DNA loops by treatment of the nucleoid with the DNA-intercalating agent ethidium bromide (80 µg/ml). Scale bars 10 µm.
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DSP027F1: Properties of naked DNA loops attached to the NM. (A) Drawing illustrating the local topology along a typical supercoiled DNA loop that correlates with both distance relative to the NM and sensitivity to DNase I. (B) Phase contrast micrograph showing the NM of a hepatocyte nucleoid. (C) Fluorescence micrograph showing the DNA halo around the NM of a hepatocyte nucleoid caused by the unwinding of the supercoiled DNA loops by treatment of the nucleoid with the DNA-intercalating agent ethidium bromide (80 µg/ml). Scale bars 10 µm.

Mentions: Our method exploits the topological properties of the average DNA loop that result from the fact that such loops are topologically constrained by being anchored to the NM, thus being equivalent to closed DNA circles. Under such a condition, the DNA molecule undergoes structural stress resulting from two factors: the covalently linked backbones of the DNA strands are helicoidal but rigid, and the low-energy hydrogen bonds between the stacked bases are quasi-statistical unions that continuously break apart and form again; such a situation poses the risk that the nucleotide bases may gyrate away from the double-helix axis and become exposed. DNA naturally solves this structural-stress problem by further coiling upon its own axis, thus avoiding the exposure of the nucleotide bases, but becoming negatively supercoiled in a similar fashion to a pulled house-telephone cord.35,36 Thus, the naked DNA loops display a gradient of supercoiling that goes from lower to higher from tip to base of the loop,36 save for the fact that the structural properties of MARs are such that they also function as buffers or sinks of negative supercoiling37,38 thus avoiding maximal supercoiling at the base of the loops. The NM plus the naked DNA loops anchored to it constitute a nucleoid. Under the conditions of lysis employed to generate nucleoids, the DNA remains essentially intact, although it lacks the nucleosome structure because of the dissociation of histones and most other nuclear proteins usually associated with DNA; yet, the DNA loops remain topologically constrained and supercoiled as depicted in Fig. 1. Indeed, nucleoids are also known as nuclear halos since the exposure of such structures to DNA-intercalating agents like ethidium bromide leads to unwinding of the DNA loops that form a DNA halo around the NM periphery (Fig. 1C). A typical DNA loop can be divided into four topological zones according to their relative proximity to the NM. Each of these zones would manifest an identifiable behaviour when exposed to non-specific nucleases that are sensitive to the local DNA topology (Fig. 1A). We have previously shown that in nucleoid preparations, the relative resistance of a given loop-DNA sequence to a limited concentration of DNase I is directly proportional to its proximity to the NM anchoring point.31,39 Two main factors determine this property. (i) Steric hindrance resulting from the proteinaceous NM that acts as a physical barrier relatively protecting the naked loop DNA that is closer to the NM from endonuclease action. (ii) The local degree of loop DNA supercoiling that is lower in the distal portions of the loop and higher in the regions proximal to the NM. Supercoiling is a structural barrier against the action of non-specific endonucleases, such as DNase I, that hydrolyze the DNA backbone by a single-strand cleavage (nicking) mechanism.40 Both factors only confer relative but not absolute DNase I resistance to loop DNA. However, in a large sample of nucleoids exposed to a limited concentration of DNase I, there is a consistent trend in which the loop-DNA sensitivity to the enzyme is inversely proportional to its distance relative to the NM and so distal regions of the loop are digested first whereas the regions closer to the NM are digested later. Indeed, it is known that the DNA embedded within the NM is very resistant to DNase I action, and there is a fraction corresponding to some 2% the total DNA that is basically non-digestible even when exposed to high concentrations of the enzyme. This fraction corresponds to fragments with an average length of 1.6 kb in rat hepatocytes,12 likely to represent the regions that include the actual MARs (LARs) anchored to the NM. This pattern of sensitivity to DNase I holds provided that the DNA is basically devoid of histones and most other proteins that form chromatin. Indeed, whole chromatin attached to the NM shows an inverse pattern of nuclease sensitivity compared with that of naked loop DNA. In looped chromatin, those sequences closer to the matrix attachment point are preferentially cleaved by nucleases.41


Determination of the in vivo structural DNA loop organization in the genomic region of the rat albumin locus by means of a topological approach.

Rivera-Mulia JC, Aranda-Anzaldo A - DNA Res. (2010)

Properties of naked DNA loops attached to the NM. (A) Drawing illustrating the local topology along a typical supercoiled DNA loop that correlates with both distance relative to the NM and sensitivity to DNase I. (B) Phase contrast micrograph showing the NM of a hepatocyte nucleoid. (C) Fluorescence micrograph showing the DNA halo around the NM of a hepatocyte nucleoid caused by the unwinding of the supercoiled DNA loops by treatment of the nucleoid with the DNA-intercalating agent ethidium bromide (80 µg/ml). Scale bars 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2818189&req=5

DSP027F1: Properties of naked DNA loops attached to the NM. (A) Drawing illustrating the local topology along a typical supercoiled DNA loop that correlates with both distance relative to the NM and sensitivity to DNase I. (B) Phase contrast micrograph showing the NM of a hepatocyte nucleoid. (C) Fluorescence micrograph showing the DNA halo around the NM of a hepatocyte nucleoid caused by the unwinding of the supercoiled DNA loops by treatment of the nucleoid with the DNA-intercalating agent ethidium bromide (80 µg/ml). Scale bars 10 µm.
Mentions: Our method exploits the topological properties of the average DNA loop that result from the fact that such loops are topologically constrained by being anchored to the NM, thus being equivalent to closed DNA circles. Under such a condition, the DNA molecule undergoes structural stress resulting from two factors: the covalently linked backbones of the DNA strands are helicoidal but rigid, and the low-energy hydrogen bonds between the stacked bases are quasi-statistical unions that continuously break apart and form again; such a situation poses the risk that the nucleotide bases may gyrate away from the double-helix axis and become exposed. DNA naturally solves this structural-stress problem by further coiling upon its own axis, thus avoiding the exposure of the nucleotide bases, but becoming negatively supercoiled in a similar fashion to a pulled house-telephone cord.35,36 Thus, the naked DNA loops display a gradient of supercoiling that goes from lower to higher from tip to base of the loop,36 save for the fact that the structural properties of MARs are such that they also function as buffers or sinks of negative supercoiling37,38 thus avoiding maximal supercoiling at the base of the loops. The NM plus the naked DNA loops anchored to it constitute a nucleoid. Under the conditions of lysis employed to generate nucleoids, the DNA remains essentially intact, although it lacks the nucleosome structure because of the dissociation of histones and most other nuclear proteins usually associated with DNA; yet, the DNA loops remain topologically constrained and supercoiled as depicted in Fig. 1. Indeed, nucleoids are also known as nuclear halos since the exposure of such structures to DNA-intercalating agents like ethidium bromide leads to unwinding of the DNA loops that form a DNA halo around the NM periphery (Fig. 1C). A typical DNA loop can be divided into four topological zones according to their relative proximity to the NM. Each of these zones would manifest an identifiable behaviour when exposed to non-specific nucleases that are sensitive to the local DNA topology (Fig. 1A). We have previously shown that in nucleoid preparations, the relative resistance of a given loop-DNA sequence to a limited concentration of DNase I is directly proportional to its proximity to the NM anchoring point.31,39 Two main factors determine this property. (i) Steric hindrance resulting from the proteinaceous NM that acts as a physical barrier relatively protecting the naked loop DNA that is closer to the NM from endonuclease action. (ii) The local degree of loop DNA supercoiling that is lower in the distal portions of the loop and higher in the regions proximal to the NM. Supercoiling is a structural barrier against the action of non-specific endonucleases, such as DNase I, that hydrolyze the DNA backbone by a single-strand cleavage (nicking) mechanism.40 Both factors only confer relative but not absolute DNase I resistance to loop DNA. However, in a large sample of nucleoids exposed to a limited concentration of DNase I, there is a consistent trend in which the loop-DNA sensitivity to the enzyme is inversely proportional to its distance relative to the NM and so distal regions of the loop are digested first whereas the regions closer to the NM are digested later. Indeed, it is known that the DNA embedded within the NM is very resistant to DNase I action, and there is a fraction corresponding to some 2% the total DNA that is basically non-digestible even when exposed to high concentrations of the enzyme. This fraction corresponds to fragments with an average length of 1.6 kb in rat hepatocytes,12 likely to represent the regions that include the actual MARs (LARs) anchored to the NM. This pattern of sensitivity to DNase I holds provided that the DNA is basically devoid of histones and most other proteins that form chromatin. Indeed, whole chromatin attached to the NM shows an inverse pattern of nuclease sensitivity compared with that of naked loop DNA. In looped chromatin, those sequences closer to the matrix attachment point are preferentially cleaved by nucleases.41

Bottom Line: Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence.The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced.We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Biología Molecular, Facultad de Medicina, Universidad Autónoma del Estado de México, Apartado Postal 428, Toluca, Edo. Méx., México.

ABSTRACT
Nuclear DNA of metazoans is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). DNA is anchored to the NM by non-coding sequences known as matrix attachment regions (MARs). There are no consensus sequences for identification of MARs and not all potential MARs are actually bound to the NM constituting loop attachment regions (LARs). Fundamental processes of nuclear physiology occur at macromolecular complexes organized on the NM; thus, the topological organization of DNA loops must be important. Here, we describe a general method for determining the structural DNA loop organization in any large genomic region with a known sequence. The method exploits the topological properties of loop DNA attached to the NM and elementary topological principles such as that points in a deformable string (DNA) can be positionally mapped relative to a position-reference invariant (NM), and from such mapping, the configuration of the string in third dimension can be deduced. Therefore, it is possible to determine the specific DNA loop configuration without previous characterization of the LARs involved. We determined in hepatocytes and B-lymphocytes of the rat the DNA loop organization of a genomic region that contains four members of the albumin gene family.

Show MeSH
Related in: MedlinePlus