Stability of proteins involved in initiation of DNA replication in UV damaged human cells.

The protein stability of the initiation factors Orc2, Orc3, Orc4, and Cdc6 was analyzed after UV light exposure in two human cell lines. In the cell line with higher repair capacity, HEK 293, no changes in the cell cycle distribution or in the protein levels of the investigated factors were detected. In HeLa cells that are characterized by lower repair capacity, UV irradiation caused a reduction of the levels of Cdc6, Orc2 and Orc3, but not of Orc4 or triggered apoptosis.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery.

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication.

The inhibitor of histone deacetylases sodium butyrate enhances the cytotoxicity of mitomycin C.

The use of histone deacetylase inhibitors has been proposed as a promising approach to increase the cell killing effect of DNA damage-inducing drugs in chemotherapy. However, the molecular mechanism of their action remains understudied. In the present article, we have assessed the effect of the histone deacetylase inhibitor sodium butyrate on the DNA damage response induced by the crosslinking agent mitomycin C.

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection.

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic.

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

Background: Histone 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.

Lack of effect of RuvB-like proteins on DNA damage signaling activation.

Ataxia telangiectasia mutated (ATM) kinase is a central player in cellular response to DNA damage. Phosphorylation of the histone H2AX by ATM is required for the accumulation of repair proteins at the sites of double-strand breaks. Recently, it was reported that the histone acetyltransferase Tat interactive protein-60 (IPP60) is required to acetylate ATM prior to its activation.

The complexity of double-strand break ends is a factor in the repair pathway choice.

The repair of double-strand breaks in mammalian cells is carried out by two pathways: homologous recombination and nonhomologous end joining. The factors that regulate the mechanism through which a specific repair pathway is activated are still not clearly defined. To study whether the complexity of the double-strand break ends is a factor that determines the choice of the repair pathway, we examined the involvement of homologous recombination by the formation of Rad51 foci in human HeLa cells treated with bleomycin and ionizing radiation.

Nuclear matrix binding site in the Rad51 recombinase.

It has been shown that the key homologous recombination protein Rad51 accumulates in DNA damage-induced nuclear foci that are attached to the nuclear matrix. In the present communication we attempted to find whether Rad51 contains a functional domain responsible for nuclear matrix binding. By alignments of the sequences encoding nuclear matrix targeting signals of human nuclear matrix binding proteins with the whole length human Rad51sequence a putative nuclear matrix targeting signal was identified.

Comparison of the global genomic and transcription-coupled repair rates of different lesions in human cells.

There are two subclasses of nucleotide excision repair (NER). One is the global genomic repair (GGR) which removes lesions throughout the genome regardless of whether any specific sequence is transcribed or not. The other is the transcription-coupled repair (TCR), which removes lesions only from the transcribed DNA sequences.

RAD51 foci formation in response to DNA damage is modulated by TIP49.

Chromatin modification plays an important role in modulating the access of homologous recombination proteins to the sites of DNA damage. TIP49 is highly conserved component of chromatin modification/remodeling complexes, but its involvement in homologous recombination repair in mammalian cells has not been examined in details. In the present communication we studied the role of TIP49 in recruitment of the key homologous recombination protein RAD51 to sites of DNA damage.

Cell cycle-dependent association of Rad51 with the nuclear matrix.

Progression of the cells through the S phase of the cell cycle is connected with accumulation of stalled and collapsed replication forks that are repaired by homologous recombination. To investigate the temporal order of homologous recombination events during the S phase, HeLa cells synchronized at the G1/S phase boundary with mimosine were released to progress into the S phase and the phosphorylation of the histone variant H2AX, the appearance of Rad51 nuclear foci and the subcellular redistribution of Rad51 were followed. The results showed that there was gradual accumulation of double-strand breaks as judged by the increase in the phosphorylation of H2AX during the S phase.

Activation of the S phase DNA damage checkpoint by mitomycin C.

We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases.

Sub-nuclear localization of Rad51 in response to DNA damage.

The repair of DNA double-strand breaks involves the accumulation of key homologous recombination proteins in nuclear foci at the sites of repair. The organization of these foci in relation to non-chromatin nuclear structures is poorly understood. To address this question, we examined the distribution of several recombination proteins in subcellular fractions following treatment of HeLa cells with ionizing radiation and the crosslinking agent mitomycin C.

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.

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.

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction.

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells.

Cells synchronized in S phase show increased rate of repair of UV damaged plasmids.

The capacity for nucleotide excision repair of cells synchronized in S phase and unsynchronized cells was compared by the host cell reactivation assay and the cell-free repair system. HeLa cells were transfected with in vitro damaged by UV irradiation pEGFP and the repair capacity was determined by the number of fluorescent cells. In the cell-free repair system, the repair capacity of protein extracts isolated from K562 cells was determined by measuring the transformation efficiency of UV irradiated pBlueScript incubated in the extracts.

DNA breaks induction by mimosine.

Mouse erythroleukemic F4 N cells were treated with mimosine, etoposide, Fe(II)-EDTA, and Cu(II) in the presence of ascorbate. DNA was isolated and subjected to agarose gel electrophoresis and the size and distribution of the DNA fragments produced by the agents were compared. With increasing concentration of Cu(II) the production of DNA fragments was increased without decrease of the average length of the fragments, and their sizes were similar to those produced by etoposide as expected for cleavage of DNA at the nuclear matrix attachments sites.

Nucleotide excision repair rates in rat tissues.

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV-irradiated pBlueScript by incubation in repair-competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair-competent extracts.

Relationship between DNA repair capacity and resistance to genotoxins in four human cell lines.

We have developed fast, reliable and simple fluorescent method to assess and compare repair capacity of cells. To this end plasmid pEGFP containing the gene for the enhanced green fluorescent protein was damaged in vitro by genotoxic agents and introduced into cells by transfection. The repair capacity of the cells was determined from the number of fluorescent cells counted with a fluorescent microscope 24 h after transfection.

DNase I sensitive site in the core region of the human beta-globin origin of replication.

HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin.

Application of the single cell gel electrophoresis on yeast cells.

In the present paper, we have applied the single cell gel electrophoresis (SCGE) assay on yeast cells treating Saccharomyces cerevisiae cells with hydrogen peroxide and methyl methanesulfonate (MMS), two DNA damaging agents. In order to overcome the problem with the yeast cell wall that prevented DNA to be extended by the electric field, we disintegrated the cell wall after embedding the cells in agarose. A characteristic picture of comets with residual nuclei and tails was observed and the length of the comet tails was dependent on the concentration of the damaging agents.

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle.

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G(1) and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-beta and the beta-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G(1) phase was enriched in origin sequences in comparison with matrix-attached DNA from early G(1) phase cells.

Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells.

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells.