Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.     

The nuclear matrix continues DNA synthesis at in vivo replicational forks

Alkaline cesium chloride gradient analysis of in vivo [3H]bromodeoxyuridine-labeled and in vitro [alpha-32P]dCTP-labeled DNA was used to determine whether in vitro DNA synthesis in regenerating rat liver nuclei and nuclear matrices continued from sites of replication initiated in vivo. At least 70 and 50% of the products of total nuclear and matrix-bound in vitro DNA synthesis, respectively, were continuations of in vivo initiated replicational forks. The relationship of the in vitro DNA synthetic sites in total nuclei versus the nuclear matrix was examined by using [3H]bromodeoxyuridine triphosphate to density label in vitro synthesized DNA in isolated nuclei and [alpha-32P]dCTP to label DNA synthesized in isolated nuclear matrix. A minimum of about 40% of matrix-bound DNA synthesis continued from sites being used in vitro by isolated nuclei. Furthermore, nuclear matrices prepared from in vitro labeled nuclei were 5-fold enriched in DNA synthesized by the nuclei and were several-fold enriched, compared to total nuclear DNA, in a particularly high density labeled population of DNA molecules.     

DNA binding properties of the nuclear matrix and individual nuclear matrix proteins. Evidence for salt-resistant DNA binding sites

The DNA binding characteristics of the rat nuclear matrix were investigated. A saturable and temperature-dependent, salt-resistant DNA binding to the nuclear matrix was discovered, with 70-80% of total bound DNA resistant to extraction with high concentrations of salt at 37 degrees C, compared to less than 5% at 0 degrees C. The initial binding of DNA to nuclear matrix is sensitive to salt concentration, indicating a transition to a salt-resistant binding state. The nuclear matrix shows a preference for single-stranded DNA, both in saturation and competition assays, with little binding of RNA or double-stranded DNA. Further competition studies show a preference for matrix-attached DNA probably involving predominantly AT-rich sequences, while a specific sequence defined previously as a matrix-attached region (MAR; Cockerill, P. N., and Garrard, W. T. (1986) Cell 46, 273-282) only showed preference for a limited number of the total matrix binding sites. These results and estimates from saturation data of approximately 150,000 single-stranded DNA binding sites per matrix lead us to propose that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity. Binding of DNA to individual matrix polypeptides separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blots was also temperature-dependent, salt-resistant, and showed a preference for binding DNA over RNA and nuclear matrix DNA over total genomic DNA. Subnuclear fractionation experiments further demonstrated that the nuclear matrix is enriched in the subset of higher molecular weight (greater than 50,000) DNA binding proteins of isolated nuclei and correspondingly depleted of the lower molecular weight ones. Of the approximately 12 major proteins separated on nonequilibrium two-dimensional gels, 7 were identified as specific DNA binding proteins including lamins A and C (but not B), and the internal nuclear matrix proteins, matrins D, E, F, G, and 4.     

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.     

Oxidative DNA damage processing in nuclear and mitochondrial DNA

Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, to examine the repair of different types of oxidative lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage.     

Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.     

Localization of AT-rich sequences at the nuclear matrix

After culture with 3H-thymidine murine erythroleukemia cells with more sites of DNA attached to the nuclear matrix have more labeled thymidine in their matrix-bound DNA than in their total DNA than do cells with fewer attachment sites. This indicates that the average attachment site sequence is enriched in A + T base pairs.     

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.     

Interaction of various nuclear matrix proteins with DNA

An attempt has been made to localize the previously detected strong and weak bonds of nuclear matrix proteins with DNA in some groups of proteins, using fractionation of the matrix into lamina and intranuclear fibrils, isolation of the "elementary globules", fractionation of matrix nucleoproteins on hydroxyapatite. It was shown that both weak and strong bonds are localized on the nuclear lamina and in the intranuclear fibrils. The single-stranded DNA enriched fraction of the matrix nucleoproteins contained mostly strong bonds. The strong bond is less resistant to pronase treatment. A method for isolating nuclear matrix nucleoprotein fractions carrying only strong or only weak bonds is proposed.     

ADP-ribosylation intensifies cleavage of DNA loops in the nuclear matrix

Using DNA pulse field electrophoresis it has been shown that ADP-ribosylation in the nucleoids of human mononuclear leukocytes and rat brain cortex neurons stimulates cleavage of DNA loops at their attachmant sites to the nuclear matrix. The conclusion has been drawn suggesting possible participation of ADP-ribosylation in DNA-topoisomerase II activity modulation in the nuclear matrix of eukaryotic cells.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.     

ADP-ribosylation intensifies cleavage of DNA loops in the nuclear matrix

Pulsed-field electrophoresis of DNA was used to demonstrate that ADP-ribosylation stimulates the cleavage of DNA loops at the sites of their anchorage to the nuclear matrix in nucleoids of human mononuclear leukocytes and rat brain neurons. ADP-ribosylation was assumed to play a role in regulating the activity of topoisomerase II in the nuclear matrix of eukaryotic cells.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 317–320.Original Russian Text Copyright © 2005 by G. Zaalishvili, Tsetskladze, Margiani, Gabriadze, T. Zaalishvili.     

The anchoring of newly synthesized adenovirus DNA to the nuclear matrix

After adenovirus infected HeLa cells were pulse labeled and pulse-chase labeled with 3H-thymidine, the nuclear matrix and DNA remaining tightly bound to the matrix were obtained by sequential cell fractionation. Measuring the radioactivity of labeled DNA indicated that newly synthesized viral DNA specifically attach to the nuclear matrix and the amount of binding DNA is in direct proportion to the viral DNA replication activity: then the DNA gradually detach from the matrix and are involved in viral assembly. Electron microscopic autoradiography of the extracted cells showed the virion and viral DNA associated with the nuclear matrix, and thus further confirmed the anchoring of newly synthesized viral DNA to the nuclear matrix.     

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.     

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.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Adenovirus DNA is associated with the nuclear matrix of infected cells.

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.     

Preferential binding of DNA primase to the nuclear matrix in HeLa cells

Studies of the spatial organization of DNA replication have provided increasing evidence of the importance of the nuclear matrix. We have previously reported a relationship between rates of DNA synthesis and the differential binding of DNA polymerase alpha to the nuclear matrix over the S-phase. We now report the detection of DNA primase bound to the HeLa nuclear matrix. Matrix-bound primase was measured both indirectly, by the incorporation of [32P]dAMP into an unprimed single-stranded template, poly(dT), and directly, by the incorporation of [3H]AMP into matrix DNA. Characteristics of this system include a requirement for ATP, inhibition by adenosine 5'-O-(thiotriphosphate), a primase inhibitor, and insensitivity to aphidicolin and alpha-amanitine, inhibitors of polymerase alpha and RNA polymerase, respectively. Subcellular quantification of primase and polymerase alpha activity revealed that while most (approximately 72%) primase activity is bound to the matrix, only a minority (approximately 32%) of polymerase alpha activity is matrix-bound. Treatment of the nuclear matrix with beta-D-octylglucoside allowed the solubilization of approximately 54% of primase activity and approximately 39% of the polymerase alpha activity. This data provides further evidence of a structural and functional role for the nuclear matrix in DNA replication. The ability to solubilize matrix-bound replicative enzymes may prove to be an important tool in the elucidation of the spatial organization of DNA replication.