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PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii. It cleaves DNA at the recognition sequence 5′-CTGCA/G-3′ generating fragments with 3′-cohesive termini[1]. This cleavage yields sticky ends 4 base pairs long. PstI is catalytically active as a dimer. The two subunits are related by a 2-fold symmetry axis which in the complex with the substrate coincides with the dyad axis of the recognition sequence. It has a molecular weight of 69,500 and contains 54 positive and 41 negatively charged residues[2].
| Recognition Sequence | Cut Site |
|---|---|
5'CTGCAG 3' 3'GACGTC 5' |
5'--CTGCA G--3' 3'--G ACGTC—5' |
The PstI restriction/modification (R/M) system has two components: a restriction enzyme that cleaves foreign DNA, and a methyltransferase which protect native DNA strands via histone methylation. The combination of both provide a defense mechanism against invading viruses[3]. The methyltransferase and endonuclease are encoded as two separate proteins and act independently. In the PstI system, the genes are encoded on opposite strands and hence must be transcribed divergently from separate promoters. The transcription initiation sites are separated by only 70 base pairs[4]. A delay in the expression of the endonuclease relative to methylase is due to the inherent differences of the two proteins[5]. The endonuclease is a dimer, requiring a second step for assembly, whereas the methylase is a monomer.
PstI is functionally equivalent to BsuBI. Both enzymes recognize the target sequence 5'CTGCAG. The enzyme systems have similar methyltransferases (41% amino acid identity), restriction endonucleases (46% amino acid identity), and genetic makeup (58% nucleotide identity)[6]. These observations suggest a shared evolutionary history.
- ^ "PstI (10 U/L) - Thermo Fisher Scientific". www.thermofisher.com. Retrieved 2016-04-29.
- ^ Walder, R. Y.; Walder, J. A.; Donelson, J. E. (1984-06-25). "The organization and complete nucleotide sequence of the PstI restriction-modification system". The Journal of Biological Chemistry. 259 (12): 8015–8026. ISSN 0021-9258. PMID 6330092.
- ^ Walder, R. Y.; Hartley, J. L.; Donelson, J. E.; Walder, J. A. (1981-03-01). "Cloning and expression of the Pst I restriction-modification system in Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America. 78 (3): 1503–1507. ISSN 0027-8424. PMC 319159. PMID 6262807.
- ^ Walder, R. Y.; Walder, J. A.; Donelson, J. E. (1984-06-25). "The organization and complete nucleotide sequence of the PstI restriction-modification system". The Journal of Biological Chemistry. 259 (12): 8015–8026. ISSN 0021-9258. PMID 6330092.
- ^ Walder, R. Y.; Walder, J. A.; Donelson, J. E. (1984-06-25). "The organization and complete nucleotide sequence of the PstI restriction-modification system". The Journal of Biological Chemistry. 259 (12): 8015–8026. ISSN 0021-9258. PMID 6330092.
- ^ Xu, G L; Kapfer, W; Walter, J; Trautner, T A (1992-12-25). "BsuBI--an isospecific restriction and modification system of PstI: characterization of the BsuBI genes and enzymes". Nucleic Acids Research. 20 (24): 6517–6523. ISSN 0305-1048. PMC 334566. PMID 1480472.