title: Characterization of MspNI (G/GWCC) and MspNII (R/GATCY), novel thermostable Type II restriction endonucleases from Meiothermus sp., isoschizomers of AvaII and BstYI.
abstract: MspNI and MspNII, isoschizomers of prototype Type II restriction endonucleases AvaII and BstYI, were extracted from an extreme thermophile bacterium belonging to the genus Meiothermus, isolated from the hot sulphur springs in north Himalayan region of India where temperature and pH ranged from 60 to 80°C and 7.5 to 8.5, respectively. The two enzymes were purified to homogeneity using Cibacron-Blue 3GA Agarose, Q-Sepharose and SP-Sepharose chromatography and were homodimers with subunit molecular weights of 27 and 45 kDa, respectively. Restriction mapping and run-off sequencing of MspNI and MspNII cleaved pBR322 DNA showed that they recognized and cleaved 5'-G/GWCC-3' and 5'-R/GATCY-3' sites, respectively. MspNI and MspNII worked optimally at 60 and 70°C, 6 and 5 mM MgCl(2), respectively and showed no star activity in organic solvents. Both were resistant to sequence methylation and were stable up to 25 PCR cycles.
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh, 160014, India.
authors.forename: Richa
authors.initials: R
authors.lastname: Gupta
authors.forename: Shuang-Yong
authors.initials: SY
authors.lastname: Xu
authors.forename: Prince
authors.initials: P
authors.lastname: Sharma
authors.forename: Neena
authors.initials: N
authors.lastname: Capalash
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008745:Methylation; D010957:Plasmids / Q000378:metabolism; D015183:Restriction Mapping; D016622:Silver Staining; D013696:Temperature; D013824:Thermus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; C056536:endodeoxyribonuclease BstYI; D015252:Deoxyribonucleases, Type II Site-Specific; C038308:GGWCC-specific type II deoxyribonucleases