Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Friedrich et al. 10.1073/pnas.0609915104.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Table 2
SI Materials and Methods




SI Figure 5

Fig. 5. Details of the mobE insertion into the phage Aeh1 nrdA gene. A portion of the DNA sequence of the Aeh1 nrdA-a/mobE/nrdA-b region is shown, with reading frames of the NrdA-a, MobE, and NrdA-b proteins indicated. The amino acid sequence of the NrdA-a protein extends from residues 415-473, and residues 1-50 for the NrdA-b protein. Amino acid residues that are conserved between the Aeh1 and T4 NrdA protein are bold and italicized, and functionally critical residues are indicated as in Fig. 1. The dashed underline indicates sequence that has no similarity to sequences in public databases. The HNH motif of mobE is indicated by a gray box. Start and stop codons are indicated for each gene, and ribosome-binding sites (RBS) are in lowercase and italicized.

Table 2. RNR-specific activities of phage and host bacterial enzymes in presence of dithiothreitol or the thioredoxin (trx) system

n.d., not determined.

*This study.

^†Berglund O, Karlström O, Reichard P (1969) Proc Natl Acad Sci USA 62:829-835.

^‡Berglund O (1972) J Biol Chem 247: 7270-7275; Berglund O, Eckstein F (1972) Eur J Biochem 28:492-496.

^§Åberg A, Hahne S, Karlsson M, Larsson A, Ormö M, Åhgren A, Sjöberg BM (1989) J Biol Chem 264:12249-12252.

^¶Kasrayan A, Birgander PL, Pappalardo L, Regnström K, Westman M, Slaby A, Gordon E, Sjöberg B-M (2004) J Biol Chem 279:31050-31057; Larsson Birgander P, Bug S, Kasrayan A, Dahlroth SL, Westman M, Gordon E, Sjöberg B-M (2005) J Biol Chem 280:14997-15003.

^||Torrents E, Westman M, Sahlin M, Sjöberg B-M (2006) J Biol Chem 281:25287-25296.

SI Materials and Methods

Plasmid Construction. For construction of expression plasmids, each phage Aeh1 gene was amplified from phage genomic DNA with gene specific primers that added restriction enzyme sites to the 5' and 3' ends of the resultant PCR products. The nrdA-a gene was amplified with primers DE-15 and DE-16 that added a 5' PciI site and a 3' BamHI site, respectively. The resultant blunt-end PCR product was cloned into pCR-Blunt, and subsequently digested with PciI/BamHI. The PciI/BamHI fragment was cloned into the NcoI/BamHI site of pACYC-Duet to yield pNrdA-a. The nrdA-b gene was amplified with primers DE-17 and DE-18 that added 5' NdeI and 3' BamHI sites, respectively, cloned into pCR-Blunt, and then into the NdeI/BamHI site of pET24a to yield pNrdA-b. To construct the dual expression plasmid pNrdA-a/NrdA-b, pNrdA-b was digested with NdeI/XhoI, and the fragment containing the nrdA-b gene was gel purified and ligated into pNrdA-a digested with NdeI/XhoI. A PCR fragment of nrdB gene was amplified with primers DE-33 and DE-34, and directly cloned into the NcoI/BamHI site of pET24d to yield pNrdB. All plasmids were sequenced to confirm that the cloned genes matched the genomic sequence.

Protein Purification. For purification of phage-encoded RNR proteins by dATPsepharose chromatography, cell pellets were resuspended in buffer A [50 mM Tris (pH 6.8)/100 mM NaCl/1 mM DTT] and protease inhibitors (Roche Diagnostics, Mississauga, Canada) added. Cells were lysed and centrifuged at 20,400 ´ g for 25 min. The supernatant was applied to a 30-ml G-25 Sephadex (GE Healthcare, Piscataway, NJ) column equilibrated in buffer A. A single 25-ml fraction was collected and applied to a 1-ml dATP-Sepharose column (Jena Bioscience) equilibrated with buffer A at 4°C. The column was washed with 20 ml of buffer A, and bound proteins were eluted with buffer A containing 1 mM dATP in 1-ml fractions. The A. hydrophila NrdA protein was purified by dATP-Sepharose chromatography directly from A. hydrophila extracts.

For purification of overexpressed NrdA-a/NrdA-b or NrdA-a/NrdA-b/NrdB complexes, dATP-Sepharose chromatography was performed as above. The peak dATPsepharose fraction was applied to a Superose 6 gel-filtration column (GE Healthcare) equilibrated in buffer A, and 0.25-ml fractions were collected at a flow rate of 0.5 ml/min. Sizes of complexes eluted from the Superose 6 column were calculated by comparison against protein standards (Sigma-Alrich, Oakville, Canada). The proportions of the proteins in the complexes were determined by PhosphorImager (GE Healthcare) analysis.

For purification of overexpressed Aeh1 NrdB, cell pellets were resuspended in 23 ml of buffer B (20 mM KHPO₄, pH 7/50 mM NaCl/2 mM DTT), lysed, and centrifuged as above. The NrdB protein was further purified by addition of 40% (wt/vol) ammonium sulfate. After dialysis, the sample was applied to a 5-ml Q-Sepharose FF column (GE Healthcare) equilibrated in buffer B, and protein was eluted with a linear gradient of NaCl to 2 M. The NrdB protein eluted at ~480 mM NaCl. Peak fractions were pooled, dialyzed against buffer B, applied to a Superose6 column equilibrated in buffer B, and eluted in 0.25-ml fractions.

Oligonucleotides. Primers for RT-PCR were DE-25 5'-TCGTTGATAACGTTAACTC G-3'; DE-26 5'-GATAGTCAAGCAAGTTATCG-3'; and DE-146 5'-CGCGAGCTCTATGTTGTTCATCATTTATTGAC-3'. PCR primers for cloning the Aeh1 nrdA-a gene were DE-15 5'-GAAGGAGACATGTTAGTAAGAAAATCAAGTGG-3'; and DE-16 5'-CGGGATCCTTAGTATCTCAGAGACTTATATTGATC-3; PCR primers for cloning the nrdA-b gene were DE-17 5'-GGAATTCCATATGATCGAACATGAAAGAATTTACG-3'; and DE-18 5'-CGGGATCCTCAGACCTTACAAACATCACAAG-3'; PCR primers for cloning the nrdB gene were DE-33 5'-CATGCCATGGATACCGTATTTAAT-3'; and DE-34 5'-CGGGATCCTTAAACTTCTAATTCTTC-3'.

• Early Edition
• Archives
• Online Submission

• Feature Articles

  (Expanded research articles of exceptional breadth)

• Commentaries

  (Expert commentary on leading research)

• Letters

  (Online-only letters to the editor)

• Inaugural Articles

  (Articles by recently elected NAS members)

• PNAS Profiles

  (Biographical profiles of Academy members)

• Sustainability Science

  (Interactions of human and environmental systems)

• Collected Papers

  (Editorials, Perspectives, Reviews, Colloquia, etc.)

• Special Features

  (Solicited articles on exceptional research topics)

• Sackler Colloquia

  (Scientific reports held under NAS auspices)