George Russev

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 104 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. J. Cell. Biochem.

Some pyrazoles as inhibitors of purine biosynthesis de novo

Abstract The inhibitory effect of some pyrazole derivatives on purine biosynthesis was studied in a pigeon liver cell-free system. It was demonstrated that 3-amino-4-carbethoxypyrazole, 3-amino-4-carboxypyrazole and 3-(3′3′-bis-β-chloroethyltriazenyl-1′)-4-carbethoxypyrazole were inhibitors, while N - β -hydroxyethyl-3-amino-4-carbethoxypyrazole was almost inactive. A possible mechanism of action is discussed.

Histone deacetylase inhibitor sodium butyrate enhances cellular radiosensitivity by inhibiting both DNA nonhomologous end joining and homologous recombination.

HDAC inhibitors have been proposed as radiosensitizers in cancer therapy. Their application would permit the use of lower radiation doses and would reduce the adverse effects of the treatment. However, the molecular mechanisms of their action remain unclear. In the present article, we have studied the radiosensitizing effect of sodium butyrate on HeLa cells. FACS analysis showed that it did not abrogate the γ-radiation imposed G2 cell cycle arrest. The dynamics of γ-H2AX foci disappearance in the presence and in the absence of butyrate, however, demonstrated that butyrate inhibited DSB repair. In an attempt to clarify which one of the two major DSBs repair pathways was affected, we synchronized HeLa cells in G1 phase and after γ-irradiation followed the repair of the DSBs by agarose gel electrophoresis. Since HR is not operational during G1 phase, by this approach we determined the rates of NHEJ only. The results showed that NHEJ decreased in the presence of butyrate. In another set of experiments, we followed the dynamics of disappearance of RAD51 foci in the presence and in the absence of butyrate after γ-radiation of HeLa cells. Since RAD51 takes part in HR only, this experiment allows the effect of butyrate on DSB repair by homologous recombination to be assessed. It showed that HR was also obstructed by butyrate. These results were confirmed by host cell reactivation assays in which the repair of plasmids containing a single DSB by NHEJ or HR was monitored. We suggest that after a DSB is formed, HDACs deacetylated core histones in the vicinity of the breaks in order to compact the chromatin structure and prevent the broken DNA ends from moving apart from each other, thus ensuring effective repair.

Mapping the Sites of Initiation of DNA Replication in Rat and Human rRNA Genes

To study the organization of DNA replication in mammalian rRNA genes, the sites of initiation of DNA synthesis in rat and human rRNA genes were mapped by two independent techniques. In rat cells the growth of the nascent DNA chains was blocked by Trioxsalen cross-links introduced in vivo. The fraction of “restricted” nascent DNA chains labeled in vivo was isolated, and the abundance in this fraction of cloned ribosomal DNA sequences was determined by hybridization. In the experiments with human cells, the nascent DNA chains were allowed to grow unrestricted for a certain period of time and the movement of the replication forks along the rRNA genes was followed by hybridization of cloned ribosomal DNA sequences to the “unrestricted” nascent DNA fragments fractionated according to size. The results show that in both rRNA genes there are two well defined regions of initiation of DNA synthesis. The first one is located upstream of the transcription units and the second one is located at the 3′-end of the coding regions of the ribosomal DNA repeats.

Nucleosome segregation in chromatin replicated in the presence of cycloheximide

Abstract Ehrlich ascites tumour cells and L cells were grown in the presence of [14C]thymidine to label DNA replicated under normal conditions and were then cultured in the presence of cycloheximide and [3H]thymidine to label DNA replicated in the absence of histone synthesis, Chromatin from these cells was digested with micrococcal nuclease and with restriction endonuclease BspRI (an isoschizomer of HaeIII). The rates of digestion of the 14C-labelled and of the 3H-labelled DNA, and the size and buoyant density of the BspRI-generated chromatin fragments showed that: (1) chromatin replicated in the presence of cycloheximide contained half the normal amount of histones; (2) it did not contain long stretches of naked DNA; and (3) it was organized in nucleosomes distributed along DNA in groups of several particles separated by relatively short stretches of histone-free DNA. Control experiments showed that this could not be the result of a long-distance sliding of nucleosomes. These data suggest a bilateral mode of nucleosome segregation during DNA replication.

Isolation of a DNA fraction from Ehrlich ascites tumour cells containing the putative origin of replication

Abstract Ehrlich ascites tumour cells were treated with Trioxsalen and long wave ultraviolet light to crosslink DNA in vivo . Despite the crosslinking the cells retained to some extent their ability to incorporate radioactive thymidine. Part of this incorporation was due to repair DNA synthesis and part to semiconservative DNA synthesis. DNA synthesized as a result of repair represented short stretches covalently linked to the high molecular weight DNA. The semiconservative DNA synthesis that had initiated between and had terminated in the vicinity of the crosslinks resulted in the formation of DNA fragments that contain the initiation site and were not ligated to the high molecular weight DNA. They were released by denaturation or by S 1 nuclease digestion in single strand or double strand form, respectively, and were separated from bulk DNA by electrophoresis or sucrose density gradient centrifugation. The reassociation analysis showed that these DNA fragments contained sequences repeated about 10 4 times per genome, which probably represented the region of the Ehrlich ascites tumour origin of replication.

Treatment of mammalian cells with mimosine generates DNA breaks.

Exponentially growing mouse erythroleukemia (MEL) cells and quiescent human peripheral blood lymphocytes (PBL) were treated with different concentrations of the nonprotein amino acid mimosine for 16 h. The treatment of the cycling cell population with 400 microM mimosine caused inhibition of DNA replication, changes in the progression of the cells in the cell cycle, and apoptosis. Nucleoid sedimentation analysis and comet assay were used to monitor the appearance and accumulation of DNA breaks. The rate of break accumulation was dose-dependent, did not depend on the stage of the cell cycle and was not connected with the mechanism of DNA replication. The data indicate that the effects of mimosine on DNA synthesis and the cell cycle may be a result of introduction of breaks into DNA.

Sodium butyrate enhances the cytotoxic effect of cisplatin by abrogating the cisplatin imposed cell cycle arrest

BackgroundHistone deacetylase inhibitors have been proposed as potential enhancers of the cytotoxic effect of cisplatin and other anticancer drugs. Their application would permit the use of lower therapeutic doses and reduction of the adverse side effects of the drugs. However, the molecular mechanisms by which they sensitize the cells towards anticancer drugs are not known in details, which is an obstacle in developing effective therapeutic protocols.ResultsIn the present work, we studied the molecular mechanisms by which sodium butyrate sensitizes cancer cells towards cisplatin. HeLa cells were treated with 5 mM butyrate, with 8 μM cis-diaminedichloroplatinum II (cisplatin), or with both. Cells treated with both agents showed approximately two-fold increase of the mortality rate in comparison with cells treated with cisplatin only. Accordingly, the life span of albino mice transfected with Ehrlich ascites tumor was prolonged almost two-fold by treatment with cisplatin and butyrate in comparison with cisplatin alone. This showed that the observed synergism of cisplatin and butyrate was not limited to specific cell lines or in vitro protocols, but was also expressed in vivo during the process of tumor development. DNA labeling and fluorescence activated cell sorting experiments showed that cisplatin treatment inhibited DNA synthesis and arrested HeLa cells at the G1/S transition and early S phase of the cell cycle. Western blotting and chromatin immunoprecipitation revealed that this effect was accompanied with a decrease of histone H4 acetylation levels. Butyrate treatment initially reversed the effect of cisplatin by increasing the levels of histone H4 acetylation in euchromatin regions responsible for the G1/S phase transition and initiation of DNA synthesis. This abrogated the cisplatin imposed cell cycle arrest and the cells traversed S phase with damaged DNA. However, this effect was transient and continued only a few hours. The long-term effect of butyrate was a massive histone acetylation in both eu- and heterochromatin, inhibition of DNA replication and apoptosis.ConclusionThe study presents evidence that cell sensitization towards cisplatin by sodium butyrate is due to hyperacetylation of histone H4 in specific chromatin regions, which temporarily abrogates the cisplatin imposed cell cycle arrest.

Organization of Plasmid DNA into Nucleosome-Like Structures after Transfection in Eukaryotic Cells

ABSTRACT One of the most often used methods to monitor DNA repair in vivo is the host cell reactivation assay (HCR). It is based on the recovery of in vitro damaged plasmids after transfection in host cells. However, it is not clear to what extent plasmid molecules were degraded in the cells and whether they were packed with histones to form chromatin. Since these questions are important to evaluate the results obtained with HCR, in the present paper we studied the fate of the plasmid pEGFP-N1 after transfection in HEK 293 cells. To this end nuclei isolated from cells transfected with native and trioxsalen crosslinked pEGFP-N1 were digested with micrococcal nuclease (MNase) and DNA was subjected to electrophoresis. Southern blots were prepared and probed with digoxigenin-labeled plasmid DNA to reveal the plasmid DNA digestion pattern. Our results showed that nucleosome-like particles were formed on both native and damaged plasmid DNA after transfection. However, the nucleosome ladders were anomalous compared to the ladders generated by digestion of bulk cellular chromatin.

DNA Repair and Cell Differentiation—Does Getting Older Means Getting Wiser as Well?

ABSTRACT The cell DNA needs to be constantly repaired in order to preserve the genome integrity and to ensure the fidelity of transcription. Its been known that a marked difference exists between rates of certain types of repair of transcribed genes and non-transcribed DNA. Recently theres been accumulating evidence that state of terminal differentiation of the cell reflects on the NER profile, attenuating ubiquitous repair in all genome regions and focusing on transcription-dependent repair. This may be viewed not as a differentiation-associated defect but, rather, as a compromise with the integrity of the genome in its bulk for the sake of exclusive repair of regions where active transcription is under way.