Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Ribosomal DNA sequences attached to the nuclear matrix

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.     

The spatial organization of DNA in the nucleus may determine positions of recombination hot-spots

We suggest a hypothesis postulating that sites of DNA loop anchorage to the nuclear matrix harbor "hot spots" of illegitimate recombination which is mediated or triggered by topoisomerase II of the nuclear matrix. Recombination between DNA loop anchorage sites may result in deletion and/or repositioning of DNA loops and loop oligomers. This hypothesis is corroborated by our own results and published results of other research groups.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

DNA distribution in fractions differing in the strength of association with nuclear matrix during activation of DNA endonucleases in cell nuclei and treatment with nuclease S1

Influence of activation of Ca2+/Mg2(+)-dependent, Mn2(+)-dependent, Mg2(+)-dependent and acidic endogenous DNAses on distribution of DNA in fractions differing in tightness of association with the nuclear matrix has been investigated. In the intact cell nuclei all types of DNA-protein bonds were obscured by a tight bonding of DNA with the proteins of replicative complex. Activation of endogenous nuclease activities caused detachment of a significant chromatin fraction from the nuclear matrix, fraction of DNA remained attached to the replicative complex, small fraction of DNA was bound to the nuclear matrix with a less tight bond. The endogenous nucleases are supposed to make cuts mainly in distal parts of the chromatin loops and do not affect the replicative complex, where the tight DNA-matrix bond is localized. Single-strand DNA-specific S1-nuclease preferably attacks the latter site.     

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix

Summary The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high lonic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area.These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.     

Secondary structure in heterogeneous nuclear RNA: involvement of regions from repeated DNA sites

Heterogeneous nuclear RNA was found to contain regions of secondary structure based on a relative resistance to nuclease treatment compared with mRNA or poliovirus RNA and a shift in density toward double-stranded RNA early in the course of nuclease digestion. The regions involved in this secondary structure are enriched for RNA segments transcribed from repeated sites in the DNA. Thus, to maximize hybridization to repetitive sites heterogeneous nuclear RNA molecules must be both denatured and fragmented. Some of the self-complementary regions in heterogeneous nuclear RNA are released by alkali denaturation and fragmentation below 1500 nucleotides but maximum release is not achieved until fragmentation below 500 nucleotides. These results indicate that these self-complementary regions (“loops” plus “stems”) are mainly below 500 nucleotides in length.     

The repair of methyl methanesulphonate-induced lesions in the DNA of a murine lymphoma cell

The introduction of single-strand breaks into the DNA of a murine lymphoma (L5178Y) cell treated in vivo with methyl methanesulphonate (MMS) and the behaviour of these breaks on post-treatment incubation were studied. A large proportion of single-strand breaks present after MMS treatment could be repaired as shown by sedimentation in alkaline sucrose. Two inhibitors of DNA synthesis, hydroxyurea and cytosine arabinoside affected the repair process differently-hydroxyurea had only a small effect while cytosine arabinoside blocked repair and at some doses allowed further degradation of the DNA. It was also found that the level of ‘repair replication’ in the presence of cytosine arabinoside was lower than that found in the presence of hydroxyurea.     

Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei.

The attachment of replicating DNA to a rapidly sedimenting nuclear structure was investigated by digestion with various nucleases. When DNA was gradually removed by DNase I, pulse label incorporated during either 1 min or during 1 hour in the presence of arabinosylcytosine, remained preferentially attached to the nuclear structure. Single strand specific digestion by nuclease S1 or staphylococcal nuclease at low concentrations caused a release of about 30% of the pulse label, without significantly affecting the attachment of randomly labelled DNA. The released material had a low sedimentation coefficient and contained most of the Okasaki fragments. The remaining pulse label was less accessible to further digestion by double strand specific nuclease activity than the bulk DNA. The results suggest that an attachment of the replication fork to the nuclear structure occurs at sites behind but close to the branch point.     

Localization of SV40 genes within supercoiled loop domains

Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.     

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimerspecific endonuclease V of bacteriophage T4. The results were consistent with the data reported by Mansbridge and Hanawalt (1983) and suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts we observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in transcribing regions of the genome.     

Initiation of unscheduled synthesis on DNA sites associated with a nuclear matrix of irradiated hepatoma cells

A study was made of the distribution of unscheduled DNA synthesis (induced by UV- or gamma-radiation and resistant to hydroxyurea) between the DNA sites in the nuclear matrix and total nuclear DNA of Zajdela ascites hepatoma cells. It was shown that during the first 1.5 to 5 min of the postirradiation incubation the rate of the unscheduled synthesis of DNA was considerably higher in the DNA fraction, firmly associated with the nuclear matrix proteins, than in the total nuclear DNA.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

Spatial Organization of DNA in the Nucleus May Determine Positions of Recombination Hot Spots

A hypothesis has been proposed that the regions of DNA loop anchorage to the nuclear matrix are the preferential sites (hot spots) of illegitimate recombination mediated or triggered by topoisomerase II of the nuclear matrix. Recombination between the regions of DNA loop anchorage to the nuclear matrix may result in deletion or repositioning of DNA loops or their groups. The proposed hypothesis is confirmed by the results of original experiments and published data obtained by other researchers.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 633–638.Original Russian Text Copyright © 2005 by Razin, Iarovaia.     

Aging and Loss of Germination in Rye Seeds Is Accompanied by a Decreased Fragmentation of Nuclear DNA at Loop Domain Boundaries

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.     

The channels model of nuclear matrix structure

The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data.     

Cell cycle-related variations in UV damage and repair capacity in chinese hamster (CHO-K1) cells

UV damage to CHO cell DNA, measured by formation of thymine-containing dimers, increases from mitosis to early S phase. Computer simulation of UV absorption by the DNA of an idealized CHO cell at different stages in the cell cycle resembles the cycle dependence of UV damage. Incision at UV damage sites, measured by the accumulation of breaks in preexisting DNA during 30 minutes' post-irradiation incubation with the DNA synthesis inhibitors 1-β-D arabinofuranosylcytosine and hydroxyurea, increases from mitosis to interphase. Analysis of the dose dependence of DNA break accumulation indicates that both the affinity of the endonuclease for dimer sites and the maximum enzyme activity at saturating levels of dimers are significantly lower in mitosis than in interphase. The killing of CHO cells by UV is enhanced if repair is temporarily inhibited by ara C. The DNA gyrase inhibitor novobiocin prevents UV-induced incision.