Identification and Characterization of the Novel Two Endonucleases Produced by Protein Splicing in Pyrococcus furiosus
Kayoko KOMORI*a, Naoki FUJITAa,b, Hideo SHINAGAWAb, Kosuke MORIKAWAc, and Yoshizumi ISHINOa
a Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), 6- 2-3 Furuedai, Suita, Osaka 565, Japan
b Department of Molecualr Microbiology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565, Japan
c Department of Structural Biology, Biomolecular Engineering Research Institute (BERI), 6- 2-3 Furuedai, Suita, Osaka 565, Japan
Inteins are initially translated as part of the precursor proteins and excised by protein-splicing. Splicing is known to proceed autocatalytically. The excised inteins are known to have site-specific endonuclease activity. Many inteins have been shown to be present in proteins from all three kingdoms, namely Bacteria, Eukarya, and Archaea, however, most of them are theoretically derived and till date, only 10 inteins are experimentally determined. Most inteins have sequence blocks resembling the LAGLI-DADG motifs of many intron encoded endonucleases, and five inteins have been experimentally demonstrated to have endonuclease activity.
We cloned the gene encoding the protein that binds specifically to Holliday- structured DNA from a genomic library of Pyrococcus furiosus. The gene encoded two putative inteins embedded in-frame within the coding region of the ribonucleotide reductase. We have subsequently determined that these inteins, named PI-Pfu I and PI-Pfu II, exhibit protein-splicing in E. coli cells and also endonuclease activity. In the presence of Mg2+ ion, they cleaved the double-stranded DNA containing their homing site (intein- allele). When Mn2+ ion was used as the cofactor instead of Mg2+ ion, another cleavage within pUC18 DNA sequence was observed. We have determined their exact cleavage sites and found that both inteins generate a 4 bp 3' -OH overhang with a 5' phosphate as other homing endonucleases. The optimal conditions of the DNA cleavage reaction including temperature, pH, concentrations of KC1 and MgC12 have also been determined.
To further our understanding of structure-function relationship, we constructed mutants having an amino acid substitution in the LAGLI-DADG motifs. Four mutants designated as 1D149A, 1E230A, 2D156A and 2D249A were isolated. All of the mutants retained the ability to bind to their recognition site, but lost the endonuclease activity.
