Histone post-translational modifications in DNA damage response.

The fact that eukaryotic DNA is packed into chromatin constitutes a physical barrier to enzymes and regulatory factors to reach the DNA molecule for replication, transcription, recombination and repair. Although most studies in this field have concentrated on how chromatin regulates transcription, there is a recent emphasis on studying the role of chromatin in the response to DNA damage. Two main chromatin-remodeling mechanisms have been identified, namely, ATP-dependent chromatin-remodeling complexes and histone post-translational modifications (PTMs). PTMs constitute reversible covalent modifications in aminoacidic residues, such as serine and threonine phosphorylation, lysine acetylation, lysine and arginine methylation and lysine ubiquitylation, among others. Moreover, nucleosome composition can be modified by the incorporation of histone variants, which are assembled into nucleosomes independently of DNA replication. The phosphorylation of the histone variant H2AX (gammaH2AX) is one of the best examples of histone PTMs in response to DNA damage induction, but many others have recently been revealed. In this review, we focus on and summarize the best-known histone PTMs observed in excision repair (base excision and nucleotide excision) and double-strand break (non-homologous end-joining and homologous recombination) repair pathways. In brief, the interplay between chromatin remodelers and DNA repair factors is discussed in relation to DNA damage response mechanisms.

Asynchronously replicating Eu/heterochromatic regions shape chromosome damage.

In order to shed more light on the influence of DNA replication on the formation and distribution of chromosome aberrations, breakpoints (BP) produced by UV-C and AluI were assigned either to the early replicating short euchromatic arm (Xp(e)) or to the late replicating long heterochromatic arm (Xq(h)) of the Chinese hamster (CHO9) X chromosome. Early (ES) or late (LS) S-phase cells were assessed by pulse incorporating the base analogue 5-bromo-2'-deoxyuridine (BrdU) immediately after UV-C irradiation (30 J/m(2)) or AluI (20 U) poration followed by BrdU immunodetection with FITC-tagged antibodies in metaphase spreads. Short (30 s) UV-C exposures (1 J/m(2)/s) induced BP preferentially in Xq(h) in LS cells and a random distribution of BP along Xp(e) and Xq(h) in ES cells. However, BP induced by long (5 min) UV-C exposures (0.1 J/m(2)/s) clustered according to arm replication time (Xp(e) during ES and Xq(h) along LS). Giemsa-stained metaphases showed elevated frequencies of UV-C induced chromatid-type aberrations and gaps, especially in cells exposed to longer UV-C irradiation. BP induced by AluI clustered in Xp(e) in ES but distributed randomly during LS. In contrast to UV-C, AluI did not produce an increase in the yield of gaps, neither in ES nor in LS cells. Putative mechanisms underlying the observed distributions of chromosome damage in replicating CHO9 cells exposed to UV-C and AluI are discussed.

Distribution of UVC-induced chromosome aberrations along the X chromosome of TCR deficient and proficient Chinese hamster cell lines.

Cells with a transcription coupled repair (TCR) deficiency are characterized by a higher sensitivity to UVC irradiation and by an increase in apoptosis and chromosomal aberration frequencies. It has been claimed that the higher frequency of chromosomal aberrations results from the transcription blockage caused by UVC-lesions located in the transcribed strands of the genome. The distribution of chromosome breakpoints in euchromatic and heterochromatic regions of the X chromosome from TCR deficient and proficient Chinese hamster cell lines was studied. Most UVC-induced breakpoints occurred in euchromatic regions of the X chromosome in both cell lines. No increase of UVC-induced breakpoints in the euchromatic region of the UV61 X chromosome was observed, indicating that TCR failure alone cannot be responsible for the increased frequency of chromosomal aberrations. Differential chromatin remodeling in the TCR defective cell line is proposed as a possible mechanism involved in the distribution of UVC-induced breakpoints along the Chinese hamster X chromosome. A similar explanation for the increase of UVC-induced chromosomal aberrations in TCR defective cells is given.

Modulation of chromosome damage localization by DNA replication timing.

PURPOSE: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xpe) and a late replicating long heterochromatic arm (Xqh). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model. MATERIALS AND METHODS: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2'-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xpe labeled) or late S-phase (Xqh labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies. RESULTS: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xpe while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xqh. CONCLUSIONS: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro: result of an IAEA-coordinated programme.

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored. It is concluded that using controlled experimentals conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.