The Arthrobacter Species FB24 Arth_1007 (DnaB) Intein Is a Pseudogene

An Arthrobacter species FB24 gene (locus tag Arth_1007) was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein). However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were ‘reverted’ back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene

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

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

DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs▿

Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex

A novel single-strand specific 3'-5' exonuclease found in the hyperthermophilic archaeon, Pyrococcus furiosus.

Nucleases play important roles in all DNA transactions, including replication, repair, and recombination. Many different nucleases from bacterial and eukaryotic organisms have been identified and functionally characterized. However, our knowledge about the nucleases from Archaea, the third domain of life, is still limited. We searched for 3'-5' exonuclease activity in the hyperthermophilic archaeon, Pyrococcus furiosus, and identified a protein with the target activity. The purified protein, encoded by PF2046, is composed of 229 amino acids with a molecular weight of 25,596, and displayed single-strand specific 3'-5' exonuclease activity. The protein, designated as PfuExo I, forms a stable trimeric complex in solution and excises the DNA at every two nucleotides from the 3' to 5' direction. The amino acid sequence of this protein is conserved only in Thermococci, one of the hyperthermophilic classes in the Euryarchaeota subdomain in Archaea. The newly discovered exonuclease lacks similarity to any other proteins with known function, including hitherto reported 3'-5' exonucleases. This novel nuclease may be involved in a DNA repair pathway conserved in the living organisms as a specific member for some hyperthermophilic archaea