Detection of PfuExo I in <i>P. furiosus</i> cells.

<p>(A) <i>P. furiosus</i> cells in the exponential growth phase (lane 1) and the stationary phase (lane 2) were harvested, and the whole cell extracts from 6×10⁸ cells, as well as 10 ng of the recombinant protein (lane 3), were subjected to 14% SDS-PAGE, followed by western blot analyses using anti-PfuExo I antiserum. (B) The recombinant proteins were boiled in the loading solution, containing 50 mM Tris-HCl, pH 6.8, 1% SDS, 1% 2-mercaptoethanol, and 10% glycerin, for 10 min (lane 1), 5 min (lane 2), and 0 min (lane 3), respectively, and subjected to 14% SDS-PAGE. The gel was stained by Coomassie Brilliant Blue.</p

Endonuclease activity of PfuExo I.

<p>Purified PfuExo I was incubated with circular ssDNA (M13 mp18) or dsDNA (pBR322) in the reaction condition described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058497#s4" target="_blank">Materials and Methods</a>. Reaction products were analyzed by electrophoresis on a 1% agarose gel, followed by ethidium bromide staining.</p

DNA cleavage activity of PfuExo I.

<p>(A) Time course experiments of the DNase activity of PfuExo I were performed, using dAC31 ssDNA labeled with ³²P at the 5′-end. PfuExo I (10 nM, as a trimer) was incubated with 5 nM DNA at 65°C. 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 [γ-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.</p

DNA binding activity of PfuExo I.

<p>Various concentrations (1, 5, 10, 50, 100, 500, or 1000 nM) of PfuExo I were incubated with ³²P-labeled ssDNA (A), dsDNA (B), 5′-overhang DNA (C), or 3′-overhang DNA (D). The protein-DNA complexes were separated by 4.5% PAGE and visualized by autoradiography.</p

Cleavage specificity of PfuExo I.

<p>Purified PfuExo I (10 nM) was incubated with 5′- overhang and 3′-overhang DNAs (5 nM) for various times at 55°C. The positions labeled by ³²P are indicated by an asterisk for each substrate. Aliquots were removed from the reactions, quenched, and then resolved by PAGE on a 12% gel containing 8 M urea.</p

Comparison of the PfuExo I amino acid sequence with those of homologs found in Thermococcaceae.

<p>Pfu, <i>P. furiosus</i>; Pho, <i>P. horikoshii</i>; Pab, <i>P. abyssi</i>; Tko, <i>T. kodakarensis</i>. Identical and similar amino acid residues are indicated by red and blue letters, respectively.</p

Purification and oligomeric state of PfuExo I.

<p>(A) The purified PfuExo I protein (2 µg) was subjected to 12% SDS-PAGE, and the gel was stained by Coomassie Brilliant Blue. Marker, molecular mass standards (New England Biolabs Inc.). (B) The oligomeric states of PfuExo I were analyzed by gel filtration. The elution profiles, monitored by the absorbance at 280 nm, are shown. The peak positions of the marker proteins from a parallel chromatographic analysis are indicated by the arrows at the top of the chromatogram. (C) The standard curve was obtained with marker proteins. The molecular weight of each marker protein is shown with the plots. The molecular weight of PfuExo I, calculated from its <i>K</i>av value, is shown.</p

Screening and identification of the gene responsible for the DNA cleavage activity.

<p>(A) The <i>E. coli</i> cells, transformed with pUC118 containing each PstI-digested DNA fragment from the positive cosmid clone, were heated. The heat-stable cells extracts from each clone were assayed for DNase activity, using ³²P-labeled d27 as the substrate. The reactions were analyzed by PAGE on a 12% gel containing 8 M urea followed by autoradiography. Lane M shows the GA ladder of d49R, generated by the Maxam-Gilbert reaction. The other lanes (1–12) are labeled d27 treated with heat-stable cell extracts from different clones. (B) The ORFs identified on the PstI-DNA fragment obtained from the positive clone #8. (C) The 5 full-length ORFs on the <i>Pst</i>I fragment in the positive clone were individually cloned and expressed in <i>E. coli</i>, and the same DNase assay was performed using ³²P-labeled d49R as the substrate. PF2046 was identified as the gene responsible for the DNA cleavage activity.</p