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. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart.

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 appearance of the truncated 49 kDa form of Cdc6 suggested the involvement of the caspase pathway in the degradation of the proteins. Reduced protein levels of Cdc6 were detected in UV damaged HeLa cells in which the apoptotic process was blocked with the caspase inhibitor Z-VAD-fmk, indicating that the degradation of Cdc6 is mediated by the proteasome pathway instead. In the presence of caffeine, an inhibitor of the cell cycle checkpoint kinases, Cdc6 was stabilized, demonstrating that its degradation is controlled by the DNA damage cell cycle checkpoint. We conclude that in response to DNA damage, the activation of origins of replication can be prevented by the degradation of Cdc6, most likely through the proteasome pathway.

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. RESULTS: In 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 microM 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. CONCLUSION: The 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.

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 RuvB-like proteins TIP48 and TIP49 are known to be necessary for the assembly and functional activity of the TIP60 acetyltransferase complex. In the present communication, we investigated the requirements of IIP48 and IIP49 for ATM activation by monitoring the cell cycle distribution and H2AX phosphorylation after irradiation of IIP48- and IIP49-depleted cells. We found that neither the cell cycle norgammay-H2AX were affected in IIP48- and IIP49-silenced cells, suggesting that the IIP60 chromatin modification complex is not engaged in DNA damage signaling upstream of ATM.

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. To prove that it is responsible for the nuclear matrix association of Rad51 18 base pairs encoding a cluster of hydrophobic amino acids in the human Rad51 Flag-tagged gene were deleted. The formation of damage-induced Rad51 foci and their association with the nuclear matrix were monitored in HeLa cells transfected with the wild-type and the mutated Rad51gene after treatment with mitomycin C. The results showed that while the wild-type protein formed Rad51 foci attached to the nuclear matrix, the mutated Rad51 failed to form DNA damage-induced nuclear foci. The loss of foci formation activity of the mutated protein was not due to impaired ability to bind double-stranded DNA in an ATP-dependent way in vitro and to bind chromatin in vivo. These data suggest that the assembly of Rad51 into nuclear foci is assisted by association with the nuclear matrix, which may support the spatial organization of the process of repair by homologous recombination.

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. The quantity of double-strand breaks was determined by gel electrophoresis and the formation of gamma-H2AX foci. Two hours after treatment with low doses of the agents that induced similar quantities of double-strand breaks that could be repaired effectively by the cells, Rad51 foci were observed only in the irradiated cells. Rad51 foci appeared in bleomycin-treated cells after prolonged exposure to the drug when the cells were arrested in the G2 phase of the cell cycle. Since bleomycin produces double-strand breaks that are less complex than the breaks induced by ionizing radiation, these results indicate that the complexity of the break ends is a factor in the choice of repair pathway and that homologous recombination is recruited in the repair of breaks with more complex multiply damaged ends during the late S and G2 phases of the cell cycle.

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. Rad51 nuclear foci did not appear until middle S phase, and this was accompanied by an increase in the chromatin- and nuclear matrix-bound Rad51 in the middle to late S phase. To determine the role of the intra S phase checkpoint in the S phase-dependent redistribution of Rad51 the cells were released in the S phase in the presence of the protein kinase inhibitors caffeine and wortmannin. The results suggest that the association of Rad51 with the nuclear matrix is regulated by activation of the intra S phase ATR-dependent checkpoint pathway.

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. In both cases, the repair capacity of the cells synchronized in S phase cells was 30-50% higher than the repair capacity of unsynchronized cells.

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. Yeast cells developed comets at concentrations at least 10 times lower than the concentrations at which comets begin to appear in mammalian cells after treatment with the two genotoxic agents. The higher sensitivity of the yeast comet assay and the fact that S. cerevisiae is one of the most thoroughly studied and easy to work with eukaryotic model system suggest that the proposed method could be an useful tool for investigation of the DNA damaging activity of potential genotoxins.

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. In contrast, mimosine and Fe(II) produced fragments of random size and with the progression of the reaction the average length of the fragments decreased. These results indicate that mimosine cuts DNA in a random fashion, regardless of its higher order chromatin organization. A conclusion is drawn that the DNA fragments obtained after mimosine treatment are a result of mimosine-assisted, Fe(II) dependent Fenton-like reactions randomly cutting chromosomal DNA.

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. There are data that GGR rates depend on the chemical nature of the lesions in a manner that the lesions inflicting larger distortion on the DNA double helix are repaired at higher rate. It is not known whether the TCR repair rates depend on the type of lesions and in what way. To address this question human cells were transfected with pEGFP and pEYFP plasmids treated with UV light, cis-diamminedichloroplatinum(II) (cisplatin) and angelicin and 24 h later the restored fluorescence was measured and used to calculate the respective NER rates. In a parallel series of experiments the same plasmids were incubated in repair-competent protein extracts to determine GGR rates in the absence of transcription. From the two sets of data, the TCR rates were calculated. We found out that cisplatin, UV light and angelicin lesions were repaired by GGR with different efficiency, which corresponded to the degree of DNA helix distortion induced by these agents. On the other hand the three lesions were repaired by TCR at very similar rates which showed that TCR efficiency was not directly connected with the chemical nature of the lesions.

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. Sodium butyrate increased mitomycin C cytotoxicity, but did not impair the repair pathways required to remove mitomycin C-induced lesions as neither the rate of nucleotide excision repair nor the homologous recombination repair rate were diminished. Sodium butyrate treatment abrogated the S-phase cell-cycle checkpoint in mitomycin C-treated cells and induced the G(2)-M checkpoint. However, sodium butyrate treatment alone resulted in accumulation of reactive oxygen species, double-strand breaks in DNA, and apoptosis. These results imply that the accumulation of reactive oxygen species-mediated increase in DNA lesion burden may be the major mechanism by which sodium butyrate enhances the cytotoxicity of 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. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

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. RAD51 redistribution to chromatin and nuclear foci formation induced by double-strand breaks and interstrand crosslinks were followed under conditions of TIP49 depletion by RNA interference. TIP49 silencing reduced RAD51 recruitment to chromatin and nuclear foci formation to about 50% of that of the control. Silencing of TIP48, which is closely related to TIP49, induced a similar reduction in RAD51 foci formation. RAD51 foci reduction in TIP49-silenced cells was not a result of defective DNA damage checkpoint signaling as judged by the normal histone H2AX phosphorylation and cell cycle distribution. Treatment with the histone deacetylase inhibitor sodium butyrate restored RAD51 foci formation in the TIP49-depleted cells. The results suggest that as a constituent of chromatin modification complexes TIP49 may facilitate the access of the repair machinery to the sites of DNA damage.

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. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.

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. The results showed association of Rad51, Rad54, BRCA1 and BRCA2, but not Rad51C, with the nuclear matrix fraction in response to double-strand breaks induction. The association of Rad51 with the nuclear matrix correlates with the formation of Rad51 nuclear foci as a result of DNA damage. Fractionation in situ confirmed that Rad51 foci remained firmly immobilized within the chromatin-depleted nuclei. Irs1SF cells that are unable to form Rad51 damage-induced nuclear foci did not show accumulation of Rad51 in the nuclear matrix. Similarly, no accumulation of Rad51 in the nuclear matrix could be observed after treatment of HeLa cells with the kinase inhibitor caffeine, which reduces formation of Rad51 foci. The results were compared to the distribution of the phosphorylated histone variant, gamma-H2AX. These data suggest a dynamic association and tethering of recombination proteins and surrounding chromatin regions to the nuclear matrix.

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. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.

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.

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.

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. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III, and subjected to hybridization with [(32)P] labeled probe located upstream of the core region of the human beta-globin replication origin. The hybridization pattern revealed the existence of a DNase I sensitive site in the core region of the beta-globin replicator. The results suggest that association with the nuclear matrix induce alteration in the chromatin structure of the origin of replication that represents a more open chromatin configuration.