Their study sometimes leads to unexpected and intriguing observations. One example is the pore-forming activity of a large group of protein toxins such as colicins A, E1, and Ia. Colicins are toxins encoded by Col-plasmids; they kill sensitive Escherichia coli strains that lack the cognate plasmid. Among the pore-forming colicins, a well-defined crystal structure was first described for colicin A. The structure of colicin A was also found to resemble Bcl-xL, which belongs to the Bcl-2 family and is a regulator of programmed cell death. This finding notably led to the discovery of the pore-forming activity of Bcl-xL. The other examples are colicin E5 and colicin D, which cleave the anticodon loops of specific transfer RNAs. Colicin E5 recognizes the QpUp sequence and mediates tRNA cleavage between positions 34 and 35 in tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp in sensitive E. coli cells. Similarly, colicin D targets 4 iso-accepting tRNA Args and cleaves between positions 38 and 39. These findings suggest that tRNA cleavage induces DNA damage. However, when D-CRD, an active domain of colicin D, was ectopically expressed in yeast cells with genetic defects in the DNA repair system, none of the cells displayed this altered sensitivity. Although the tRNA species cleaved by D-CRD are different from those cleaved by zyomcin and PaT, this result suggested that DNA damage is not an indirect consequence of tRNA cleavage, and we tried to elucidate the mechanism by which PaT Orf2p induces DNA damage. We then found evidence that PaT Orf2p cleaves DNA, in addition to its specific tRNA cleavage activity. Moreover, the translation impairment caused by tRNA cleavage allows AbMole Etidronate translocation of Orf2p into the nucleus, leading to histone phosphorylation. Orf2p needs to translocate into the nucleus to interact with genomic DNA. A previous study has shown that green fluorescent protein -tagged Orf2p expression localizes in the cytosol, but not in the nucleus. Nevertheless, GFP tagging may change Orf2p localization. Our previous report has shown that Orf2p expression AbMole Indinavir sulfate impairs translation in host cells and induces a similar transcriptional response as the treatment with cycloheximide. This translational inhibition decreases Orf2p expression, thus complicating detection by western blotting. Orf2p-H299A was then expressed, and the cells were treated with cycloheximide to mimic the translation inhibition induced by tRNA cleavage. After treatment, cell fractionation was carried out, and the localization of Orf2pH299A in the nucleus was examined by western blotting. Notably, Orf2p levels in the nucleus increased, although total Orf2p was slightly decreased after cycloheximide treatment. The results suggested that decreased translation activity permits Orf2p to translocate into the nucleus and cleave DNA. Although PaT reportedly induces DNA fragmentation, the mechanism and correlation with tRNA cleavage had not yet been proven. This study showed that Orf2p, a subunit of PaT responsible for tRNA cleavage, cleaves chromosomal DNA. His299, which is required for tRNA cleavage, is also involved in DNA cleavage. His residues are also found in other DNases, such as DNase I. Our results indicate that tRNA cleavage alone does not induce a DNA damage response, because the histone is not phosphorylated by the expression of D-CRD or ��subunit. As mentioned suggesting that these repair systems respond to direct DNA cleavage of PaT Orf2p. Conversely, the reason DNA repair is required to compete with zymocin remains unclear. In addition to cleaving tRNA, zymocin reportedly changes the multiple aspects of the physiological status of sensitive yeast cells, which may be involved in the DNA repair process.