''Pst''I is a type II

restriction endonuclease A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...

isolated from the Gram negative species, '' Providencia stuartii''.

Function

''Pst''I cleaves DNA at the recognition sequence 5′-CTGCA/G-3′ generating fragments with 3′-cohesive termini. This cleavage yields

sticky ends DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the ...

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 A recognition sequence is a DNA sequence to which a structural motif of a DNA-binding domain exhibits binding specificity. Recognition sequences are palindromes. The transcription factor Sp1 for example, binds the sequences 5'-(G/T)GGGCGG(G/A)(G/ ...

. It has a molecular weight of 69,500 and contains 54 positive and 41 negatively charged residues. The PstI restriction/modification (R/M) system has two components: a restriction enzyme that cleaves foreign DNA, and a

methyltransferase Methyltransferases are a large group of enzymes that all methylate their substrates but can be split into several subclasses based on their structural features. The most common class of methyltransferases is class I, all of which contain a Rossm ...

which protect native DNA strands by methylation of the adenine base inside the recognition sequence. The combination of both provide is a defense mechanism against invading viruses. 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 pair A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...

s. A delay in the expression of the endonuclease relative to methylase is due to the inherent differences of the two proteins. 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). These observations suggest a shared

evolution Evolution is change in the heritable characteristics of biological populations over successive generations. These characteristics are the expressions of genes, which are passed on from parent to offspring during reproduction. Variation ...

ary history. When examining the preferential double strand cleavage of DNA, the restriction endonuclease PstI bind to pSM1 plasmid DNA.

DNA cloning

PstI is a useful enzyme for DNA cloning as it provides a selective system for generating hybrid DNA molecules. These hybrid DNA molecules can be then cleaved at the regenerated PstI sites. Its use is not limited to molecular cloning; it is also used in restriction site mapping, genotyping, Southern blotting, restriction fragment length polymorphism (RFLP) and SNP. It is also an

isoschizomer Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other. The first enzyme discovered which recognizes a given sequence is known as th ...

restriction enzyme SalPI from ''Streptomyces albus P''.

Cleavage

PstI preferentially cleaves purified pSM1 DNA without being influenced by the superhelicity of the substrate. However, it is not known whether the effects of this cleavage occurs upon binding to the recognition site or DNA scission. Its differential cleavage rates at different restriction sites is due to the five features of duplex structure. The proximity to the ends in linear DNA molecule, variation in DNA sequence within the recognition sites for enzymes, short distance between regions of unusual DNA sequences and recognition sites, and lastly the special structures such as loops and hairpins. The collective effect of these five factors could affect the accessibility of the restriction enzyme to its recognition sites.

Relation

References

{{restriction enzyme Molecular biology Restriction enzymes