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.     

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.     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system. J. Cell. Biochem. 70:596–603. © 1998 Wiley-Liss, Inc.     

Participation of Poly(ADP-ribose)-polymerase of nuclear matrix in DNA repair

The poly(ADP-ribose)-polymerase activity of brain and liver cell nuclei is changed during X-irradiation of rats. In the nuclear matrix, poly(ADP-ribose)-polymerase activity increases at a low dose of irradiation (1.7 Gy) and decreases at a high dose (6.7 Gy). A significant part of the activity of nuclear NMN-adenylyltransferase, a key enzyme for biosynthesis of NAD (the substrate of poly(ADP-ribose)-polymerase), has been found in the nuclear matrix. An interrelation between ADP-ribosylation taking place on the matrix level and eukaryotic cell DNA repair is suggested.     

ADP-ribosylation of the nuclear matrix in Physarum polycephalum

Less than 10% of the total ADP-ribosylation in isolated nuclei of Physarum polycephalum are bound to the nuclear matrix. In S-phase the matrix-associated ADP-ribosylation is almost twice as high as compared with the G2-period of the cell cycle. Inhibitors of DNA- and RNA-synthesis and the mutagen N-methyl-N′-nitro-N-nitrosoguanidine increase the percentage of matrix-associated ADP-ribosylation.     

Release of template restriction for DNA synthesis by poly ADP(ribose) polymerase during the HeLa cell cycle.

The degree of complexing between DNA and chromosomal proteins and the ability of poly adenosine diphosphate ribosylation (ADP-ribosylation) of nuclear proteins to release this template restriction and expose DNA primer site changes during the HeLa cell cycle. Primer site exposure by NAD and poly ADP(ribose) polymerase was assessed with intact nuclei by single deoxynucleotide incorporation into DNA in the presence of saturating bacterial DNA polymerase. The most marked in vitro enhancement of primer site exposure by ADP-ribosylation occurred in early G1 phase, where cellular template restriction was the greatest. Cytoplasmic DNA polymerase also had high activity in early G1 phase of the cell cycle. Streptozotocin reduces NAD pools in HeLa cells; a concomitant stimulation of nuclear poly ADP(ribose) polymerase activity is noted.     

TARG1 protects against toxic DNA ADP-ribosylation

ADP-ribosylation is a modification that targets a variety of macromolecules and regulates a diverse array of important cellular processes. ADP-ribosylation is catalysed by ADP-ribosyltransferases and reversed by ADP-ribosylhydrolases. Recently, an ADP-ribosyltransferase toxin termed ‘DarT’ from bacteria, which is distantly related to human PARPs, was shown to modify thymidine in single-stranded DNA in a sequence specific manner. The antitoxin of DarT is the macrodomain containing ADP-ribosylhydrolase DarG, which shares striking structural homology with the human ADP-ribosylhydrolase TARG1. Here, we show that TARG1, like DarG, can reverse thymidine-linked DNA ADP-ribosylation. We find that TARG1-deficient human cells are extremely sensitive to DNA ADP-ribosylation. Furthermore, we also demonstrate the first detection of reversible ADP-ribosylation on genomic DNA in vivo from human cells. Collectively, our results elucidate the impact of DNA ADP-ribosylation in human cells and provides a molecular toolkit for future studies into this largely unknown facet of ADP-ribosylation.     

Changes in the level of poly ADP-ribosylation during a cell cycle

In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.     

ADP-ribosylation of nuclear proteins in the rat brain

The distribution of (ADP-ribose)n synthesized from [14C]NAD labeled at the adenyl ring in several protein fractions of isolated rat brain nuclei was studied. Preferential ADP-ribosylation of nonhistone nuclear proteins was shown to occur. It was demonstrated that pol (ADP-ribose)polymerase and DNA-topoisomerase II are located spatially close to each other. A correlation between ADP-ribosylation and the activity of nuclear matrix DNA-topoisomerase II was established.     

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.     

Regulatory potential of S/MAR elements in transient expression

S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from a previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 77–81.Original Russian Text Copyright © 2005 by Sass, Ruda, Akopov, Snezhkov, Nikolaev, Sverdlov.     

The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).     

Poly(adenosine diphosphate-ribosylation) of nuclear matrix proteins in alkylating agent resistant and sensitive cell lines

Using Walker 256 breast carcinoma cell lines either with or without acquired resistance to alkylating agents, the structural framework proteins of the nucleus, the nuclear matrix proteins, were found to be effective acceptors for poly(ADP-ribose). Incubation of isolated nuclei with nicotinamide adenine [32P] dinucleotide ([32P] NAD), followed by the isolation of the nuclear matrix, demonstrated that two polypeptides of approximate molecular weight (Mr) 105 000 and 116 000 were extensively poly(ADP-ribosylated). By an in vitro [32P] NAD assay, the nuclear matrix fraction was found to maintain approx. 15% of the total nuclear matrix activity of poly(ADP-ribose) polymerase. Confirmation that the trichloroacetic acid (TCA) precipitable material represented ADP-ribose units was achieved by enzymatic digestion of the nuclear matrix preparation with snake venom phosphodiesterase (SVP). Within 15 min, greater than 85% of the 32P label was digested by SVP and the final digestion products were found to be phosphoribosyl-AMP (PR-AMP) and adenosine 5'-monophosphate (5'-AMP) by thin layer chromatographic analysis. The average polymer chain length was estimated to be 6-7 ADP-ribose units. Because poly(ADP-ribose) polymerase has a putative role in DNA repair, a comparison of the nuclear matrix fractions from Walker resistant and sensitive tumor cell lines was made. In both cell lines, the quantitative and qualitative patterns of the nuclear matrix associated poly(ADP-ribosylation) were similar.     

Study of nuclear poly(ADP-ribose)polymerase and DNA-topoisomerase II of brain cells during postnatal development of rats

The nuclear poly(ADP-ribose)polymerase activity of neuronal and glial cells during postnatal development of rats was studied. It was shown that the poly(ADP-ribose)polymerase activity of nuclei and nuclear matrix of neuronal cells during postnatal development of rats is increased, whereas the polymerase activity of glial cell nuclei and nuclear matrix in newborn and adult rats is higher than in 14-day-old animals. The DNA-topoisomerase II activity of neuronal nuclear matrix during the postnatal development of rats does not change, whereas the topoisomerase activity of glial nuclear matrix decreases but is always higher than the DNA-topoisomerase II activity of neuronal cell matrix during the postnatal development of rats. It is suggested that ADP-ribosylation in the nuclear matrix of neuronal cells causes the inhibition of the DNA-topoisomerase II activity of nuclear matrix.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.