Chromatin remodelling and chromosome damage distribution.

Histone acetylation/deacetylation constitute the most relevant chromatin remodelling mechanism to control DNA access to nuclear machinery as well as to mutagenic agents. Thus, these epigenetics mechanisms could be involved in processing DNA lesions into chromosomal aberrations. Although radiation-induced DNA lesions are believed to occur randomly, in most cases chromosome breakpoints appear distributed in a non-random manner. In order to study the distribution of chromosome damage induced by clastogenic agents in relation to chromosome histone acetylation patterns, an experimental model based on treating Chinese hamster cells with endonucleases and ionizing radiations as well as immunolabelling metaphase chromosomes with antibodies to acetylated histone H4 was developed. The analysis of intra- and interchromosome breakpoint distribution has been carried out on G-banded chromosomes, and results obtained were correlated with chromosome acetylated histone H4 profiles. A co-localization of intrachromosomal breakpoints induced by Alu I, Barn HI and DNase I as well as by neutrons and gamma-rays was observed. Radiation- and endonuclease-induced breakpoints tend to cluster in less condensed chromosome regions (G-light bands) that show the highest levels of acetylated histone H4. The analysis of interchromosomal distribution of radiation-induced lesions showed a concentration ofbreakpoints in Chinese hamster chromosomes with particular histone acetylation patterns. The fact that chromosome break-points occur more frequently in transcriptionally competent chromosome regions suggests that chromatin conformation and nuclear architecture could play a role in the distribution of chromosome lesions.

Distinct mechanisms of nonhomologous end joining in the repair of site-directed chromosomal breaks with noncomplementary and complementary ends.

DNA double-strand breaks (DSBs) are considered the most important type of DNA damage inflicted by ionizing radiation. The molecular mechanisms of DSB repair by nonhomologous end joining (NHEJ) have not been well studied in live mammalian cells, due in part to the lack of suitable chromosomal repair assays. We previously introduced a novel plasmid-based assay to monitor NHEJ of site-directed chromosomal I-SceI breaks. In the current study, we expanded the analysis of chromosomal NHEJ products in murine fibroblasts to focus on the error-prone rejoining of DSBs with noncomplementary ends, which may serve as a model for radiation damage repair. We found that noncomplementary ends were efficiently repaired using microhomologies of 1-2 nucleotides (nt) present in the single-stranded overhangs, thereby keeping repair-associated end degradation to a minimum (2-3 nt). Microhomology-mediated end joining was disrupted by Wortmannin, a known inhibitor of DNA-PKcs. However, Wortmannin did not significantly impair the proficiency of end joining. In contrast to noncomplementary ends, the rejoining of cohesive ends showed only a minor dependence on microhomologies but produced fivefold larger deletions than the repair of noncomplementary ends. Together, these data suggest the presence of several distinct NHEJ mechanisms in live cells, which are characterized by the degree of sequence deletion and microhomology use. Our NHEJ assay should prove a useful system to further elucidate the genetic determinants and molecular mechanisms of site-directed DSBs in living cells.

Checking your breaks: surveillance mechanisms of meiotic recombination.

Numerous DNA double-strand breaks (DSBs) are introduced into the genome in the course of meiotic recombination. This poses a significant hazard to the genomic integrity of the cell. Studies in a number of organisms have unveiled the existence of surveillance mechanisms or checkpoints that couple the formation and repair of DSBs to cell cycle progression. Through these mechanisms, aberrant meiocytes are delayed in their meiotic progression, thereby facilitating repair of meiotic DSBs, or are culled through programmed cell death, thereby protecting the germline from aneuploidies that could lead to spontaneous abortions, birth defects and cancer predisposition in the offspring. Here we summarize recent progress in our understanding of these checkpoints. This review focuses on the surveillance mechanisms of the budding yeast S. cerevisiae, where the molecular details are best understood, but will frequently compare and contrast these mechanisms with observations in other organisms.

Imaging of protein movement induced by chromosomal breakage: tiny 'local' lesions pose great 'global' challenges.

Interruption of chromosomal integrity by DNA double-strand breaks (DSBs) causes a major threat to genomic stability. Despite tremendous progress in understanding the genetic and biochemical aspects of DSB-induced genome surveillance and repair mechanisms, little is known about organization of these molecular pathways in space and time. Here, we outline the key spatio-temporal problems associated with DSBs and focus on the imaging approaches to visualize the dynamics of DSB-induced responses in mammalian cells. We delineate benefits and limitations of these assays and highlight the key recent discoveries where live microscopy provided unprecedented insights into how cells defend themselves against genome-destabilizing effects of DNA damage.

Cell cycle-dependent regulation of double-strand break repair: a role for the CDK.

DNA Double-Strand Breaks (DSBs) are dangerous lesions that can lead to genomic instability and to cell death. Eukaryotic cells repair DSBs either by nonhomologous end joining (NHEJ) or by homologous recombination (HR). Recent work has allowed to study the ability of yeast cells to repair a single, chromosomal DSB, at different stages of the cell cycle. Yeast cells repair the broken chromosome during the G(1) stage only by NHEJ, whereas HR is the mechanism of choice during the rest of the cell cycle. HR does not require duplicated chromatids or passage through S-phase. Control over the fate of the broken chromosome is exerted by Clb-CDK activity, which is required to carry out the first step of HR, ssDNA resection. Similar results in other organisms suggest that this control is a conserved feature in all eukaryotes.

The control of Spo11's interaction with meiotic recombination hotspots.

Programmed double-strand breaks (DSBs), which initiate meiotic recombination, arise through the activity of the evolutionary conserved topoisomerase homolog Spo11. Spo11 is believed to catalyze the DNA cleavage reaction in the initial step of DSB formation, while at least a further 11 factors assist in Saccharomyces cerevisiae. Using chromatin-immunoprecipitation (ChIP), we detected the transient, noncovalent association of Spo11 with meiotic hotspots in wild-type cells. The establishment of this association requires Rec102, Rec104, and Rec114, while the timely removal of Spo11 from chromatin depends on several factors, including Mei4 and Ndt80. In addition, at least one further component, namely, Red1, is responsible for locally restricting Spo11's interaction to the core region of the hotspot. In chromosome spreads, we observed meiosis-specific Spo11-Myc foci, independent of DSB formation, from leptotene until pachytene. In both rad50S and com1Delta/sae2Delta mutants, we observed a novel reaction intermediate between Spo11 and hotspots, which leads to the detection of full-length hotspot DNA by ChIP in the absence of artificial cross-linking. Although this DNA does not contain a break, its recovery requires Spo11's catalytic residue Y135. We propose that detection of uncross-linked full-length hotspot DNA is only possible during the reversible stage of the Spo11 cleavage reaction, in which rad50S and com1Delta/sae2Delta mutants transiently arrest.

DNA repair: keeping it together.

A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.

Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex.

Chromosome aberrations are common outcomes of exposure to DNA-damaging agents or altered replication events and are associated with various diseases and a variety of carcinomas, including leukemias, lymphomas, sarcomas, and epithelial tumors. The incidence of aberrations can be greatly increased as a result of defects in DNA repair pathways. Although there is considerable information about the molecular events associated with the induction and repair of a double-strand break (DSB), little is known about the events that ultimately lead to translocations or deletions through the formation of chromosome breaks or the dissociation of broken ends. We describe a system for visualizing DNA ends at the site of a DSB in living cells. After induction of the break, DNA ends flanking the DSB site in wild-type cells remained adjacent. Loss of a functional RMX complex (Rad50/Mre11/Xrs2) or a mutation in the Rad50 Zn-hook structure resulted in DNA ends being dispersed in approximately 10%-20% of cells. Thus, the RMX complex holds broken ends together and counteracts mitotic spindle forces that can be destructive to damaged chromosomes.

DNA breaks promote genomic instability by impeding proper chromosome segregation.

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.

Spontaneous cell transformation: karyoplasts derived from multinucleated cells produce new cell growth in senescent human epithelial cell cultures.

Previously, it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells was a dynamic process of nuclear and cellular events. In this process, nuclei of polyploid (above 2N) cells broke down into multinucleated cells (MNCs) by amitotic division. An induced mass karyoplast (i.e., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended life span (EL) and altered morphology. In this study, without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis). These cell transformation events were followed by phase-contrast photography of living cell cultures. Primary, diploid, epithelial cell cultures grew for two to three passages and then entered senescence. Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume. Some of these cells were MNCs, mostly with two to four nuclei. Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed, and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over one to two passages. At the end of their replicative phase, the EL cells entered another death crisis (M2) from which no cells survived. In M2-crisis, rarely transformed cells appear with immortal cell growth characteristics (i.e., cell lines). Numerous examples of fragmentation or amitosis of polyploid nuclei in the production of multinucleated cells (MNCs) are presented. Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations. The nuclear and cellular events in cell transformation are compared with a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas. The possibility of a connection between these two processes is discussed. And finally the difference in the duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared with that in senescence MNCs.

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage.

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.

Fission yeast Sap1 protein is essential for chromosome stability.

Sap1 is a dimeric sequence-specific DNA binding-protein, initially identified for its role in mating-type switching of the fission yeast Schizosaccharomyces pombe. The protein is relatively abundant, around 10,000 dimers/cell, and is localized in the nucleus. sap1+ is essential for viability, and transient overexpression is accompanied by rapid cell death, without an apparent checkpoint response and independently of mating-type switching. Time lapse video microscopy of living cells revealed that the loss of viability is accompanied by abnormal mitosis and chromosome fragmentation. Overexpression of the C terminus of Sap1 induces minichromosome loss associated with the "cut" phenotype (uncoupling mitosis and cytokinesis). These phenotypes are favored when the C terminus of Sap1 is overexpressed during DNA replication. Fluorescence in situ hybridization experiments demonstrated that the cut phenotype is related to precocious centromere separation, a typical marker for loss of cohesion. We propose that Sap1 is an architectural chromatin-associated protein, required for chromosome organization.

Radiation-induced genomic instability and its implications for radiation carcinogenesis.

Radiation-induced genomic instability is characterized by an increased rate of genetic alterations including cytogenetic rearrangements, mutations, gene amplifications, transformation and cell death in the progeny of irradiated cells multiple generations after the initial insult. Chromosomal rearrangements are the best-characterized end point of radiation-induced genomic instability, and many of the rearrangements described are similar to those found in human cancers. Chromosome breakage syndromes are defined by chromosome instability, and individuals with these diseases are cancer prone. Consequently, chromosomal instability as a phenotype may underlie some fraction of those changes leading to cancer. Here we attempt to relate current knowledge regarding radiation-induced chromosome instability with the emerging molecular information on the chromosome breakage syndromes. The goal is to understand how genetic and epigenetic factors might influence the onset of chromosome instability and the role of chromosomal instability in carcinogenesis.

Characterization of a knock-out mutation at the Gc2 locus in wheat.

Gametocidal (Gc) genes, introduced into common wheat from related Aegilops species, are selfish genetic elements that ensure their preferential transmission by inducing chromosomal breaks. Here we report the production and characterization of a knock-out mutation of the Gc2 gene transferred to wheat as a wheat-Aegilops sharonensis T4B-4S(sh)#1 translocation chromosome. In hemizygous Gc2/- condition, gametophytes lacking Gc2 suffer chromosomal fragmentation and produce non-functional gametes, which leads to sporophytic semisterility and exclusive transmission of the Gc2-carrier chromosome. We have identified one putative ethyl methylsulfonate (EMS)-induced Gc2 mutant that restores spike fertility and shows Mendelian segregation. Progeny screening mapped the mutation to the Gc2-carrier chromosome T4B-4S(sh)#1. C-banding and fluorescence in situ hybridization analyses showed that the loss of Gc2 function in the mutant is not due to a terminal deficiency. Analysis of first and second pollen mitoses in Gc2(mut) /- plants and C-banding analysis of testcross progenies showed that no chromosomal breakage occurs in the mutant. No gametophytic chromosomal breakage was observed in heterozygous Gc2(mut) /Gc2 plants, which had fully fertile spikes. These results suggest that Gc2 encodes two agents, one causing chromosomal breaks in gametophytes lacking Gc2 and another that protects the Gc2 carrier from breakage. The EMS-induced Gc2 mutant appears to be a knock-out of the gene encoding the "breaking" agent. These data are a first crucial step toward the molecular understanding of Gc2 action.

The Fanconi anaemia/BRCA pathway.

Fanconi anaemia (FA) is a rare genetic cancer-susceptibility syndrome that is characterized by congenital abnormalities, bone-marrow failure and cellular sensitivity to DNA crosslinking agents. Seven FA-associated genes have recently been cloned, and their products were found to interact with well-known DNA-damage-response proteins, including BRCA1, ATM and NBS1. The FA proteins could therefore be involved in the cell-cycle checkpoint and DNA-repair pathways. Recent studies implicate the FA proteins in the process of repairing chromosome defects that occur during homologous recombination, and disruption of the FA genes results in chromosome instability--a common feature of many human cancers.

Role of oxidative stress in telomere shortening in cultured fibroblasts from normal individuals and patients with ataxia-telangiectasia.

Cells from patients with the autosomal recessive disorder ataxia-telangiectasia (A-T) display accelerated telomere shortening upon culture in vitro. It has been suggested that A-T cells are in a chronic state of oxidative stress, which could contribute to their enhanced telomere shortening. In order to examine this hypothesis, we monitored the changes in telomere length in A-T homozygous, heterozygous and control fibroblasts cultured in vitro under various conditions of oxidative stress using quantitative fluorescent in situ hybridization. Compared with normal cells, the rate of telomere shortening was 1.5-fold increased under 'normal' levels of oxidative stress in A-T heterozygous cells and 2.4-3.2-fold in A-T homozygous cells. Mild chronic oxidative stress induced by hydrogen peroxide increased the rate of telomere shortening in A-T cells but not in normal fibroblasts and the telomere shortening rate decreased in both normal and A-T fibroblasts if cultures were supplemented with the anti-oxidant phenyl-butyl-nitrone. Increased telomere shortening upon oxidative stress in A-T cells was associated with a significant increase in the number of extra-chromosomal fragments of telomeric DNA and chromosome ends without detectable telomere repeats. We propose that the ATM (A-T mutated) protein has a role in the prevention or repair of oxidative damage to telomeric DNA and that enhanced sensitivity of telomeric DNA to oxidative damage in A-T cells results in accelerated telomere shortening and chromosomal instability.

In vitro cleavage of the MLL gene by topoisomerase II inhibitor (etoposide) in normal cord and peripheral blood mononuclear cells.

The correlation between infant leukemia and in utero exposure to topoisomerase II (topo-II) inhibitor has been clarified. We examined the in vitro effect of topo-II inhibitor (etoposide) on cleavage of the MLL gene in cord and peripheral blood mononuclear cells (MNCs). Southern blot analysis showed cleavage of the MLL gene in peripheral blood MNCs of infants when the MNCs were exposed to etoposide. MNCs were incubated with etoposide at various concentrations (1 to 50 microM), and a ligation-mediated polymerase chain reaction (LM-PCR) was used to detect double strand breaks (DSBs) of DNA in intron 8 of the MLL breakpoint cluster region. PCR products obtained with LM-PCR were subcloned and sequenced to identify the breakpoint in the MLL gene. The PCR products indicated DSBs of the MLL gene were obtained without any difference in the incidence between 3 different samples (cord and peripheral blood from infants and children). Sequencing analysis showed that the DSBs occurred on the telomeric side of intron 8 and near exon 9. There was no evidence that the cord blood was more susceptible to MLL DNA breakage by topo-II inhibitor than were other cells. Instability of the partner gene during the fetal period could be associated with the pathogenesis of infant leukemia.

Chromatin remodeling during spermiogenesis: a possible role for the transition proteins in DNA strand break repair.

An important chromatin remodeling process is taking place during spermiogenesis in mammals and DNA strand breaks must be produced to allow the accompanying change in DNA topology. Endogenous DNA strand breaks are indeed detected at mid-spermiogenesis steps but are no longer present in mature sperm. Both in vitro and in vivo evidence suggests that the DNA-binding and condensing activities of a set of basic nuclear "transition proteins" may be crucial to the integrity of the chromatin remodeling process. We propose that these proteins are necessary for the repair of the strand breaks so that DNA fragmentation is minimized in the mature sperm.

Probabilities of radiation-induced inter- and intrachromosomal exchanges and their dependence on the DNA content of the chromosome.

A biophysical model has been developed that is based on the assumptions that an interphase chromosome occupies a spherical territory and that chromosome exchanges are formed by the misrejoining of two DNA double-strand breaks induced within a defined interaction distance. The model is used to explain the relative frequencies of inter- and intrachromosomal exchanges and the relationship between radiation-induced aberrations in individual chromosomes and the DNA content of the chromosome. Although this simple model predicts a higher ratio of inter- to intrachromosomal exchanges for low-LET radiation than for high-LET radiation, as has been suggested by others, we argue that the comparison of the prediction of the model with experimental results is not straightforward. With the model, we also show that the probability of the formation of interchromosomal exchanges is proportional to the "surface area" of the chromosome domain plus a correction term. The correction term is small if the interaction distance is less than 1 microm for both low- and high-LET radiations.

Genomic integrity and the repair of double-strand DNA breaks.

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents or as intermediates in normal cellular processes, creates a severe threat for the integrity of the genome. Unrepaired or incorrectly repaired DSBs lead to broken chromosomes and/or gross chromosomal rearrangements which are frequently associated with tumor formation in mammals. To maintain the integrity of the genome and to prevent the formation of chromosomal aberrations, several pathways exist in eukaryotes: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). These mechanisms are conserved in evolution, but the relative contribution depends on the organism, cell type and stage of the cell cycle. In yeast, DSBs are primarily repaired via HR while in higher eukaryotes, both HR and NHEJ are important. In mammals, defects in both HR or NHEJ lead to a predisposition to cancer and at the cellular level, the frequency of chromosomal aberrations is increased. This review summarizes our current knowledge about DSB-repair with emphasis on recent progress in understanding the precise biochemical activities of individual proteins involved.