Ver 100uC under anaerobic conditions, and its DNA transactions have been partially characterized [9,16,17]. The exposure of P. furiosus cells to ionizing radiation reportedly resulted in chromosomal fragmentation, but a following incubation of the cells at 95uC resulted in chromosome reassembly [14]. These findings suggested that P. furiosus must have a highly MedChemExpress hPTH (1-34) efficient DNA repair system for DNA strand breaks, but the details about the components and mechanism of the system still remain unknown. Deoxyribonucleases (DNases) operate as central components of most DNA repair systems, by executing or initiating DNA damage removal to promote cell survival and ensure genetic integrity [18,19]. DNases can be classified into structure-, damage-, or sequence-specific families, with respect to their substrate preferences. They can also be divided into exonucleases, which hydrolyze nucleic acids from either the 59 or 39 end, and endonucleases, which hydrolyze internal phosphodiester bonds without the requirement of a free DNA end. Many exonucleases have been identified in Eukarya and Bacteria [18], and their functions have been analyzed both in vitro and in vivo. Singlestranded DNA specific exonucleases are particularly abundant in E. coli, where they play important roles in several repair processes within the cell, such as MMR [4], HR [20], and tandem repeat stabilization [21]. Although similar functional exonucleases areIdentification of Novel Nuclease from P. furiosusexpected to be involved in the repair systems in P. furiosus, no single-stranded DNA specific 39?9 exonuclease has been identified yet, except for the DNA polymerase-associated exonuclease activity [22,23]. To understand the DNA repair systems in more detail in the hyperthermophilic archaea, the unknown factors, including DNases, must be identified. In this study, we identified a novel nuclease in P. furiosus cells. This enzyme degrades single-stranded DNA, but not doublestranded DNA, from the 39 
to 59 direction. No conserved sequence motifs for the 39?9 exonucleases were detected in the MedChemExpress Licochalcone-A deduced amino acid sequence of this protein. Furthermore, genes encoding the homologous sequence of this enzyme are present only in the Thermococci in Archaea annotated as hypothetical protein in the public databases, and therefore, it is a novel nuclease.Results Identification of a Nuclease Activity in the Heat-stable Protein Library from P. furiosusWe searched the databases for an ORF bearing a sequence homologous to those of the known 39?9 exonucleases in the P. furiosus genome, to detect homologs of known exonucleases. However, no homologous sequence was found. Therefore, we tried to identify a 18204824 novel enzyme containing a 39?9 exonuclease by screening for the activity from the heat-stable protein libraries derived from P. furiosus, as described in the Materials and Methods section [24]. Among 500 independent heat extracts of E. coli clones transformed by the cosmid-based gene library of P. furiosus, we isolated a clone producing a protein that degrades the synthetic oligonucleotide. The cosmid DNA containing a P. furiosus genomic DNA of 35 kbp was recovered from the E. coli clone exhibiting the activity. The inserted genomic DNA fragment was subcloned into the pUC118 plasmid vector after PstI digestion. Then, as a second screening, we searched for the nuclease activity in the heatresistant cell extract from each transformant. One clone with the target activity was found (lane 8 in Fig. 1A). The pl.Ver 100uC under anaerobic conditions, and its DNA transactions have been partially characterized [9,16,17]. The exposure of P. furiosus cells to ionizing radiation reportedly resulted in chromosomal fragmentation, but a following incubation of the cells at 95uC resulted in chromosome reassembly [14]. These findings suggested that P. furiosus must have a highly efficient DNA repair system for DNA strand breaks, but the details about the components and mechanism of the system still remain unknown. Deoxyribonucleases (DNases) operate as central components of most DNA repair systems, by executing or initiating DNA damage removal to promote cell survival and ensure genetic integrity [18,19]. DNases can be classified into structure-, damage-, or sequence-specific families, with respect to their substrate preferences. They can also be divided into exonucleases, which hydrolyze nucleic acids from either the 59 or 39 end, and endonucleases, which hydrolyze internal phosphodiester bonds without the requirement of a free DNA end. Many exonucleases have been identified in Eukarya and Bacteria [18], and their functions have been analyzed both in vitro and in vivo. Singlestranded DNA specific exonucleases are particularly abundant in E. coli, where they play important roles in several repair processes within the cell, such as MMR [4], HR [20], and tandem repeat stabilization [21]. Although similar functional exonucleases areIdentification of Novel Nuclease from P. furiosusexpected to be involved in the repair systems in P. furiosus, no single-stranded DNA specific 39?9 exonuclease has been identified yet, except for the DNA polymerase-associated exonuclease activity [22,23]. To understand the DNA repair systems in more detail in the hyperthermophilic archaea, the unknown factors, including DNases, must be identified. In this study, we identified a novel nuclease in P. furiosus cells. This enzyme degrades single-stranded DNA, but not doublestranded DNA, from the 39 to 59 direction. No conserved sequence motifs for the 39?9 exonucleases were detected in the deduced amino acid sequence of this protein. Furthermore, genes encoding the homologous sequence of this enzyme are present only in the Thermococci in Archaea annotated as hypothetical protein in the public databases, and therefore, it is a novel nuclease.Results Identification of a Nuclease Activity in the Heat-stable Protein Library from P. furiosusWe searched the databases for an ORF bearing a sequence homologous to those of the 
known 39?9 exonucleases in the P. furiosus genome, to detect homologs of known exonucleases. However, no homologous sequence was found. Therefore, we tried to identify a 18204824 novel enzyme containing a 39?9 exonuclease by screening for the activity from the heat-stable protein libraries derived from P. furiosus, as described in the Materials and Methods section [24]. Among 500 independent heat extracts of E. coli clones transformed by the cosmid-based gene library of P. furiosus, we isolated a clone producing a protein that degrades the synthetic oligonucleotide. The cosmid DNA containing a P. furiosus genomic DNA of 35 kbp was recovered from the E. coli clone exhibiting the activity. The inserted genomic DNA fragment was subcloned into the pUC118 plasmid vector after PstI digestion. Then, as a second screening, we searched for the nuclease activity in the heatresistant cell extract from each transformant. One clone with the target activity was found (lane 8 in Fig. 1A). The pl.