Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.Chromatin reorganization by histone modification and mobilization plays a crucial role in DNA metabolism, including replication, transcription, and repair. It appears that histone modification and mobilization can reorganize chromatin to allow DNA repair machinery to access damaged chromosomal DNA (11,29,52,56,57).H2AX is a histone variant that differs from H2A at various amino acid residues along the entire protein and in its Cterminal extensions. H2AX is phosphorylated after the induction of DNA double-strand breaks (DSBs), and the phosphorylated H2AX (␥-H2AX) participates in focus formation at sites of DNA damage. After induction of DSBs, the MRN complex (MRE11, RAD50, and NBS1) binds to broken DNA ends and recruits active ATM, ATR, and/or DNA protein kinase, resulting in the initial phosphorylation of H2AX (32,38,40). MDC1 then associates with ␥-H2AX and recruits additional activated ATM to the sites of DSBs (23,46). This positive feedback loop leads to the expansion of the ␥-H2AX region surrounding DSBs and provides docking sites for many DNA damage and repair proteins, including the MRN complex, 53BP1, and BRCA1 (5, 6, 46). ␥-H2AX plays a role in the accumulation but not in the initial recruitment of repair factors such as the MRN complex, 53BP1, and BRCA1 (10, 63). Therefore, modifications of H2AX other than phosphorylation could play a role in the initial step of the DNA damage response.Until recently, the biological significance of ubiquitination in the DNA damage response has been unclear. H2B ubiquitination regulates the damage checkpoint response (15). H2A is ubiquitinated during the response to UV-induced DNA damage (8). UV-induced DNA damage also causes the ubiquitination of histones H3 and H4, resulting in their release from chromatin (60). Interestingly, ubiquitin-conjugated proteins appear to be accumulated at sites of DSBs, forming nuclear foci like ␥-H2AX (34). These findings raise the possibility that histone ubiquitination is also involved in the reorganization of chromatin in response to D...

Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G 2 /M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTB-GFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48 -124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.Cytolethal distending toxin (CDT) 1 is a unique bacterial toxin that induces cell cycle arrest of cultured cells in the G 2 phase. It has been identified in several pathogenic bacteria including Campylobacter spp., Escherichia coli, Shigella dysenteriae, Haemophilus ducreyi, Helicobacter hepaticus, and Actinobacillus actinomycetemcomitans. The cells intoxicated with CDT show a cytopathic effect and distension in cell size, and eventually they die, which has been shown to be a common consequence of CDT treated cells (1-4). CDT holotoxin is composed of CDTA, -B, and -C, encoded by the cdtA, cdtB, and cdtC genes tandemly located on the cdt locus (1, 5-7). The CDTinduced G 2 arrest has been ascribed to the inactivation of the Cdc2-cyclin B complex, which is a key molecule for the progression of the cell cycle. In normal cells, dephosphorylation of the Thr-14 and Tyr-15 in Cdc2 triggers G 2 /M transition in the cell cycle. CDT-treated cells were found to maintain Cdc2 with these residues phosphorylated in the Cdc2-cyclin B complex (8). This is because of the recruitment of Cdc25C, a Cdc2-specific phosphatase, from the nucleus to cytoplasm, which prevents dephosphor...

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Cytolethal distending toxin (CDT) is a newly identified virulence factor produced by several pathogenic bacteria implicated in chronic infection. Seventy three strains of periodontopathogenic bacteria were examined for the production of CDT by a HeLa cell bioassay and for the presence of the cdt gene by PCR with degenerative oligonucleotide primers, which were designed based on various regions of the Escherichia coli and Campylobacter cdtB genes, which have been successfully used for the identification and cloning of cdtABC genes from Actinobacillus actinomycetemcomitans Y4 (M. Sugai et al., Infect. Immun. 66:5008-5019, 1998). CDT activity was found in culture supernatants of 40 of 45 tested A. actinomycetemcomintans strains, but the titer of the toxin varied considerably among these strains. PCR experiments indicated the presence of Y4-type cdt sequences in these strains, but the rest of A. actinomycetemcomitans were negative by PCR amplification and also by Southern blot analysis for the cdtABC gene. In the 40 CDT-positive strains, Southern hybridization with HindIII-digested genomic DNA revealed that there are at least 6 restriction fragment length polymorphism types. This suggests that the cdtABC flanking region is highly polymorphic, which may partly explain the variability of the CDT activity in the culture supernatants. The rest of tested strains of periodontopathogenic bacteria did not have detectable CDT production by the HeLa cell assay and for cdtB sequences by PCR analysis under our experimental conditions. These results strongly suggested that CDT is a unique toxin predominantly produced by A. actinomycetemcomitans among periodontopathogenic bacteria.Periodontitis is a destructive inflammatory response affecting the tooth-supporting tissues. Etiological and microbiological studies have well established that dental plaque, a composite of microorganisms and their products, plays a major role in the pathogenesis of periodontitis (2, 34). Previous evidence suggests that it participates in promoting inflammation of gingival tissue through direct cytotoxicity and indirect immunemediated responses (33). A variety of bacterial products in dental plaque have been implicated in this process. Actinobacillus actinomycetemcomitans has been suspected to be one of the key pathogens in the etiology of human periodontitis (30,34). It produces a variety of virulence factors including cytotoxic factors (2, 8-12, 17, 19, 28, 31), chemotactic inhibitors (33), collagenases (24), and lipopolysaccharides (13, 25). Among the cytotoxic factors, leukotoxin has been the most extensively studied (14-16, 18). Recently, we and others discovered another cytotoxic factor which shows cell cycle-specific growth inhibitory activity as a new member of the cytolethal distending toxin (CDT) family in A. actinomycetemcomitans Y4 (18, 28, 32). The CDTs are produced by a variety of bacterial genera and form a heterogeneous family of toxins with similar biological activities (4,20,23,26). The term CDT was designated for an activity that ind...

Clinical Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT) with titers ranging from 102 to 108 U/mg. Single nucleotide polymorphism analysis of the cdt gene in clinical isolates identified a variation of a single amino acid at residue 281 of CdtB, which significantly affected CDT toxicity by modulating the chromatin-degrading activity of CdtB.