Obably occurs because BIBS39 supplier PfuExo I needs to grasp a 4? nt-long DNA strand to express its nucleolytic activity. To obtain further evidence that PfuExo I degrades the singlestranded DNA in the 39 to 59 direction, the nuclease assays were performed using 39 and 59- overhang DNAs. As shown in Fig. 5, the reaction products were detected only from the 39- overhang DNA substrate, and the cleavage reaction stopped at 
the 30-mer. This is the position of the 3rd nucleotide from the end of the double-stranded region. This result supports the proposal that PfuExo I is an exonuclease with 39 to 59 polarity, and its substrate DNA must be at least a few nucleotides long for PfuExo I to bind. It is interesting to analyze the degradation mode of the PfuExo I from the enzymological aspect. We did several experiments investigating how processive this enzyme is for its cleavage of the DNA strand. Constant PfuExo I and increasing amounts of substrate DNA were reacted. The cleavage pattern showed that the amounts of 25331948 DNA strands with shorter sizes decreased with theIdentification of Novel Nuclease from P. furiosusFigure 4. DNA cleavage activity of PfuExo I. (A) Time course experiments of the DNase activity of PfuExo I were performed, using dAC31 ssDNA labeled with 32P at the MedChemExpress 86168-78-7 59-end. PfuExo I (10 nM, as a trimer) was incubated with 5 nM DNA at 65uC. For each time point, aliquots were removed from the reaction and quenched. These samples were subjected to PAGE on an 18 gel containing 8M urea, and the degradation products 
were visualized by autoradiography using TyphoonTrio (GE Healthcare). The 7-nt, 10-nt, and 14-nt oligonucleotides with the same sequence as dAC31, and [c-32P]ATP were loaded alongside to provide markers. (B) dA30, dC30, and dT30 were used as substrates with 5 nM of PfuExo I. The samples were subjected to PAGE on a 12 gel containing 8M urea, and the degradation products were visualized by autoradiography. The 10-nt, 15-nt, and 20-nt poly dAs were loaded alongside to provide markers. doi:10.1371/journal.pone.0058497.gincreasing amounts of substrate DNA (data not shown). This result and other experiments suggested that the cleavage mode of PfuExo I is distributive.Examination of the Endonuclease Activity of PfuExo ITo determine whether PfuExo I also has endonuclease activity, endonuclease assays were performed using 10457188 the circular forms of single- and double-stranded DNAs. As shown in Fig. 6, no cleavage was detected with either ssDNA or dsDNA, even after a 30-min incubation. This result indicated that PfuExo I requires the terminus to express the nuclease activity.only one band was observed (Fig. 7C, and 7D), suggesting that the preferential binding of PfuExo I occurred at the single-stranded region, which is not long enough for the second binding. These observations are consistent with the results of the nuclease assays, showing the single-stranded specific cleavage. Various lengths of ssDNAs were used for the gel shift assays and distinct shift band was observed from the ssDNA of 7 nt-long (data not shown), supporting that 7 nt-long is sufficient for PfuExo I to stably grasp DNA.PfuExo I Homologs in ArchaeaThe deduced amino acid sequence of PfuExo I is not similar to any proteins from eukaryotic and bacterial organisms in the public databases. From a comprehensive search of the public databases, ORFs with highly similar sequences were detected only from the organisms that belong to the Thermococcaceae (6 Pyrococcus and 10 Thermococcus species) in t.Obably occurs because PfuExo I needs to grasp a 4? nt-long DNA strand to express its nucleolytic activity. To obtain further evidence that PfuExo I degrades the singlestranded DNA in the 39 to 59 direction, the nuclease assays were performed using 39 and 59- overhang DNAs. As shown in Fig. 5, the reaction products were detected only from the 39- overhang DNA substrate, and the cleavage reaction stopped at the 30-mer. This is the position of the 3rd nucleotide from the end of the double-stranded region. This result supports the proposal that PfuExo I is an exonuclease with 39 to 59 polarity, and its substrate DNA must be at least a few nucleotides long for PfuExo I to bind. It is interesting to analyze the degradation mode of the PfuExo I from the enzymological aspect. We did several experiments investigating how processive this enzyme is for its cleavage of the DNA strand. Constant PfuExo I and increasing amounts of substrate DNA were reacted. The cleavage pattern showed that the amounts of 25331948 DNA strands with shorter sizes decreased with theIdentification of Novel Nuclease from P. furiosusFigure 4. DNA cleavage activity of PfuExo I. (A) Time course experiments of the DNase activity of PfuExo I were performed, using dAC31 ssDNA labeled with 32P at the 59-end. PfuExo I (10 nM, as a trimer) was incubated with 5 nM DNA at 65uC. For each time point, aliquots were removed from the reaction and quenched. These samples were subjected to PAGE on an 18 gel containing 8M urea, and the degradation products were visualized by autoradiography using TyphoonTrio (GE Healthcare). The 7-nt, 10-nt, and 14-nt oligonucleotides with the same sequence as dAC31, and [c-32P]ATP were loaded alongside to provide markers. (B) dA30, dC30, and dT30 were used as substrates with 5 nM of PfuExo I. The samples were subjected to PAGE on a 12 gel containing 8M urea, and the degradation products were visualized by autoradiography. The 10-nt, 15-nt, and 20-nt poly dAs were loaded alongside to provide markers. doi:10.1371/journal.pone.0058497.gincreasing amounts of substrate DNA (data not shown). This result and other experiments suggested that the cleavage mode of PfuExo I is distributive.Examination of the Endonuclease Activity of PfuExo ITo determine whether PfuExo I also has endonuclease activity, endonuclease assays were performed using 10457188 the circular forms of single- and double-stranded DNAs. As shown in Fig. 6, no cleavage was detected with either ssDNA or dsDNA, even after a 30-min incubation. This result indicated that PfuExo I requires the terminus to express the nuclease activity.only one band was observed (Fig. 7C, and 7D), suggesting that the preferential binding of PfuExo I occurred at the single-stranded region, which is not long enough for the second binding. These observations are consistent with the results of the nuclease assays, showing the single-stranded specific cleavage. Various lengths of ssDNAs were used for the gel shift assays and distinct shift band was observed from the ssDNA of 7 nt-long (data not shown), supporting that 7 nt-long is sufficient for PfuExo I to stably grasp DNA.PfuExo I Homologs in ArchaeaThe deduced amino acid sequence of PfuExo I is not similar to any proteins from eukaryotic and bacterial organisms in the public databases. From a comprehensive search of the public databases, ORFs with highly similar sequences were detected only from the organisms that belong to the Thermococcaceae (6 Pyrococcus and 10 Thermococcus species) in t.
