Chapter 6. Application of new methods for detection of DNA damage and repair.

New methods for detecting DNA damage and repair are reviewed and their potential significance is discussed. These include methods based on analysis of DNA damage-induced chromatin modifications, cytological detection of DNA repair synthesis, damage-induced immobilization of repair proteins and living cell imaging. Special attention is paid to current methods of detection of modifications of histones and other proteins associated with DNA double-strand breaks which represent most dangerous genome damage. New methods of analysis of DNA damage and repair may be useful in biodosimetry, early cancer diagnostics and in the analysis of efficiency of cancer radiation therapy and chemotherapy.

Regulation of mammalian gene expression by retroelements and non-coding tandem repeats.

Genomes of higher eukaryotes contain abundant non-coding repeated sequences whose overall biological impact is unclear. They comprise two categories. The first consists of retrotransposon-derived elements. These are three major families of retroelements (LINEs, SINEs and LTRs). SINEs are clustered in gene-rich regions and are found in promoters of genes while LINEs are concentrated in gene-poor regions and are depleted from promoters. The second class consists of non-coding tandem repeats (satellite DNAs and TTAGGG arrays), which are associated with mammalian centromeres, heterochromatin and telomeres. Terminal TTAGGG arrays are involved in telomere capping and satellite DNAs are located in heterochromatin, which is implicated in transcription silencing by gene repositioning (relocalization). It is unknown whether interstitial TTAGGG sequences, which are present in many vertebrates, have a function. Here, evidence will be presented that retroelements and TTAGGG arrays are involved in regulation of gene expression. Retroelements can provide binding sites for transcription factors and protect promoter CpG islands from repressive chromatin modifications, and may be also involved in nuclear compartmentalization of transcriptionally active and inactive domains. Interstitial telomere-like sequences can form dynamically maintained three-dimensional nuclear networks of transcriptionally inactive domains, which may be involved in transcription silencing like classic heterochromatin.

Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells.

Double-strand DNA breaks (DSBs) induced by ionizing radiation can be visualized in human cells using antibodies against Ser-139 phosphorylated histone H2AX (gamma-H2AX). Large gamma-H2AX foci are seen in the nucleus fixed 1 hour after irradiation and their number corresponds to the number of DSBs, allowing analysis of these genome lesions after low doses. We estimated whether transcription is affected in chromatin domains containing gamma-H2AX by following in vivo incorporation of 5-bromouridine triphosphate (BrUTP) loaded by cell scratching (run-on assay). We found that BrUTP incorporation is strongly suppressed at gamma-H2AX foci, suggesting that H2AX phosphorylation inhibits transcription. This is not caused by preferential association of gamma-H2AX foci with constitutive or facultative heterochromatin, which was visualized in irradiated cells using antibodies against histone H3 trimethylated at lysine-9 (H3-K9m3) or histone H3 trimethylated at lysine-27 (H3-K27m3). Apparently, formation of gamma-H2AX induces changes of chromatin that inhibit assembly of transcription complexes without heterochromatin formation. Inhibition of transcription by phosphorylation of histone H2AX can decrease chromatin movement at DSBs and frequency of misjoining of DNA ends.

Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells.

Sensitivity of tumor cells to treatment with anticancer drugs depends on expression and function of antiapoptotic and antioxidant proteins. The goal of our study was to determine the functional role of the novel antioxidant protein Peroxiredoxin V (PrxV), in protection of human lung carcinoma cell lines against apoptosis. Analysis of expression of PrxV in multiple lung carcinoma cell lines revealed a positive correlation between the expression of PrxV and radioresistance in vitro. Clones of the lung carcinoma cells U1810 with down-regulated expression of PrxV, or with its impaired enzymatic function (expression of redox-negative PrxV), demonstrated increased sensitivity to treatment with anticancer drugs etoposide and adriamycin. Pre-treatment of these clones with antioxidant N-acetyl-cysteine did not change their sensitivity to adriamycin, suggesting the involvement of a non-redox activity of PrxV. Expression of the redox-negative PrxV mainly affected the mitochondrial pathway of apoptosis, as assessed by cytochrome c release assay. Impairment of the PrxV enzymatic function also affected transmembrane potential and calcium loading capacity of mitochondria, as well as mitochondrial morphology. Altogether, these findings suggest that PrxV is a multifunctional protein, which is essential for protection against apoptosis induced by anticancer drugs.

Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.

Clusters of regulatory signals for RNA polymerase II transcription associated with Alu family repeats and CpG islands in human promoters.

Primate genomes contain a very large number of short interspersed GC-rich repeats of the Alu family, which are abundant in introns and intergenic spacers but also present in 5' flanking regions of genes enriched in binding motifs (BMs) for transcription factors and frequently containing CpG islands. Here we studied whether CpG islands located in promoters of human genes overlap with Alu repeats and with clusters of BMs for the zinc-finger transcription factors Sp1, estrogen receptor alpha, and YY1. The presence of estrogen-response elements in Alu was shown earlier and here we confirm the presence in the consensus Alu sequence of the binding sites for Sp1 and YY1. Analyzing >5000 promoters from the two databases we found that Alu sequences are underrepresented in promoters compared to introns and that approximately 4% of CpG islands located within the -1000 to +200 segments of human promoters overlap with Alu repeats. Although this fraction was found to be lower for proximal segments of promoters (-500 to +100), our results indicate that a significant number (>1000) of all human genes may be controlled by Alu-associated CpG islands. Analysis of clustering of potential BMs for the indicated transcription factors within some promoters also suggests that the Alu family contributed to the evolution of transcription cis-regulatory modules in the human genome. It is important that among Alu sequences overlapping with CpG islands in promoters a large fraction of members of the old Alu subfamilies is found, suggesting extensive retroposon-assisted regulatory genome evolution during the divergence of the primates.

Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus.

The Ser-139 phosphorylated form of replacement histone H2AX (gamma-H2AX) is induced within large chromatin domains by double-strand DNA breaks (DSBs) in mammalian chromosomes. This modification is known to be important for the maintenance of chromosome stability. However, the mechanism of gamma-H2AX formation at DSBs and its subsequent elimination during DSB repair remains unknown. gamma-H2AX formation and elimination could occur by direct phosphorylation and dephosphorylation of H2AX in situ in the chromatin. Alternatively, H2AX molecules could be phosphorylated freely in the nucleus, diffuse into chromatin regions containing DSBs and then diffuse out after DNA repair. In this study we show that free histone H2AX can be efficiently phosphorylated in vitro by nuclear extracts and that free gamma-H2AX can be dephosphorylated in vitro by the mammalian protein phosphatase 1-alpha. We made N-terminal fusion constructs of H2AX with green fluorescent protein (GFP) and studied their diffusional mobility in transient and stable cell transfections. In the absence or presence of DSBs, only a small fraction of GFP-H2AX is redistributed after photobleaching, indicating that in vivo this histone is essentially immobile in chromatin. This suggests that gamma-H2AX formation in chromatin is unlikely to occur by diffusion of free histone and gamma-H2AX dephosphorylation may involve the mammalian protein phosphatase 1alpha.

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization.

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.

End-joining of reconstituted histone H2AX-containing chromatin in vitro by soluble nuclear proteins from human cells.

Non-homologous end-joining is an important pathway for the repair of DNA double-strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser-139 in the histone variant H2AX to form gamma-H2AX. Here we report efficient in vitro end-joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end-joining is not suppressed by the PI-3 kinase inhibitor wortmannin. During the end-joining reaction H2AX is phosphorylated at Ser-139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end-joining reaction appears to be independent of H2AX phosphorylation.