A new aspect of a restriction endonuclease Tth111 I. It has a degenerated specificity (Tth111 I).
We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II. The former does not cleave phi X174RFDNA and the latter does. We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T. thermophilus 111. The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively. However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule. The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I*. The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains. The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al. The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites).
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; C027716:endodeoxyribonuclease Tth111II; D015252:Deoxyribonucleases, Type II Site-Specific