Mutations in the Arsp-FB24 Arth_1007 intein fail to restore activity.

<p>Only unspliced MIP precursor (109 kDa) was observed with the wild type intein or with inteins mutated at essential intein residues that restored the consensus amino acid at position B∶10 (Asn⁶⁵) or possible F∶4 positions (Asp³¹⁵ or Gly³¹¹). Simply Blue Safe stained SDS-PAGE of soluble lysates after in vivo expression at 37°C (0) or after incubation in vitro at 37°C at the indicated pH values in the presence (+) or absence (−) of 50 mM DTT. Western Blots with anti-P sera failed to detect spliced MP or cleavage products in all samples (data not shown). Molecular weight standards are in the left lane of each gel and selected sizes are listed in kDa (New England Biolabs 10 to 250 kDa ladder).</p

Class 3 inteins are monophyletic.

<p>A phylogenetic tree of Class 3 inteins based on conserved intein motifs was generated using MrBayes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Huelsenbeck1" target="_blank">[15]</a> in the Geneious software package. The scale bar represents 0.3 substitutions per site. A larger phylogenetic analysis previously excluded all non-Class 3 inteins examined from this clade <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Tori2" target="_blank">[11]</a>. Except for the Arsp-FB-24 Arth_1007 intein, intein names are defined in the InBase database (<a href="http://www.neb.com/neb/inteins.html" target="_blank">http://www.neb.com/neb/inteins.html</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Perler2" target="_blank">[6]</a> with the additional T3 suffix, which indicates that these are Class 3 inteins.</p

Sequence alignment of the Arsp-FB24 Arth_1007 intein (Arsp) vs. the MP-Catera Gp206 intein (Catera).

<p>Conserved splicing motifs (A, B, F, and G) and homing endonuclease motifs (C, D, E and H), as described in the InBase intein database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Perler1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Pietrokovski1" target="_blank">[3]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Pietrokovski2" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026361#pone.0026361-Perler2" target="_blank">[6]</a>, are indicated above the Arsp-FB24 Arth_1007 intein sequence. Positions within each motif are numbered from amino to carboxy terminal and are referred to using the motif letter and the position number separated by a colon. Arsp-FB24 Arth_1007 intein residues Asn⁶⁵ in Motif B position 10 (B∶10) and Gly³¹¹ in Motif F position 4 (F∶4) are underlined. Residues present in both inteins are listed and similar substitutions are marked with a plus sign.</p

The native Arsp-FB24 Arth_1007 precursor in inactive.

<p>The native Arsp-FB24 Arth_1007 precursor (NIC) was expressed in E.coli with the addition of an N-terminal His tag. Because the C-extein is only 14 residues, a precursor truncated at the intein C-terminus (NI) was also expressed. The left panel is a SDS-PAGE of soluble lysates after in vivo expression at 37°C stained with Simply Blue Safe Stain and the right panel is a Western blot of the same samples run in the same gel. Proteins containing the N-terminal His tag were detected with the anti-His tag antibody. The control lane (CTR) contains soluble lysates from E.coli with just an empty plasmid. The sizes of the molecular weight standards (M) are listed in kDa (New England Biolabs 10 to 250 kDa ladder).</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

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

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

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

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

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