PstI

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PstI is a type II restriction endonuclease isolated from the Gram negative species, Providencia stuartii .

Contents

Function

PstI 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 SequenceCut 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 by methylation of the adenine base inside the recognition sequence. The combination of both provide is 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.

When examining the preferential double strand cleavage of DNA, the restriction endonuclease PstI bind to pSM1 plasmid DNA. [7]

DNA cloning

PstI is a useful enzyme for DNA cloning as it provides a selective system for generating hybrid DNA molecules. [8] 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. [9] It is also an isoschizomer restriction enzyme SalPI from Streptomyces albus P. [10]

Cleavage

PstI preferentially cleaves purified pSM1 DNA without being influenced by the superhelicity of the substrate. [11] 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.

Related Research Articles

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase 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 of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

The restriction modification system is found in bacteria and archaea, and provides a defense against foreign DNA, such as that borne by bacteriophages.

In molecular biology, endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically, while many, typically called restriction endonucleases or restriction enzymes, cleave only at very specific nucleotide sequences. Endonucleases differ from exonucleases, which cleave the ends of recognition sequences instead of the middle (endo) portion. Some enzymes known as "exo-endonucleases", however, are not limited to either nuclease function, displaying qualities that are both endo- and exo-like. Evidence suggests that endonuclease activity experiences a lag compared to exonuclease activity.

<i>Fok</i>I Restriction enzyme

The restriction endonuclease Fok1, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non sequence-specific DNA cleavage domain at the C-terminal. Once the protein is bound to duplex DNA via its DNA-binding domain at the 5'-GGATG-3' recognition site, the DNA cleavage domain is activated and cleaves the DNA at two locations, regardless of the nucleotide sequence at the cut site. The DNA is cut 9 nucleotides downstream of the motif on the forward strand, and 13 nucleotides downstream of the motif on the reverse strand, producing two sticky ends with 4-bp overhangs.

<i>Hin</i>dIII Enzyme

HindIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis.

<i>Hae</i>III Enzyme

HaeIII is one of many restriction enzymes (endonucleases) a type of prokaryotic DNA that protects organisms from unknown, foreign DNA. It is a restriction enzyme used in molecular biology laboratories. It was the third endonuclease to be isolated from the Haemophilus aegyptius bacteria. The enzyme's recognition site—the place where it cuts DNA molecules—is the GGCC nucleotide sequence which means it cleaves DNA at the site 5′-GG/CC-3. The recognition site is usually around 4-8 bps.This enzyme's gene has been sequenced and cloned. This is done to make DNA fragments in blunt ends. HaeIII is not effective for single stranded DNA cleavage.

<i>Eco</i>RV Restriction enzyme

EcoRV is a type II restriction endonuclease isolated from certain strains of Escherichia coli. It has the alternative name Eco32I.

<span class="mw-page-title-main">Neoschizomer</span> Type of restriction enzymes

Neoschizomers are restriction enzymes that recognize the same nucleotide sequence, but cleave at different sites. The first restriction enzyme discovered to recognize a sequence is called the prototype, and others that recognize the same sequence are isoschizomers. Neoschizomers are a subset of isoschizomers.

Deoxyribonuclease IV (phage-T4-induced) is catalyzes the degradation nucleotides in DsDNA by attacking the 5'-terminal end.

Arthrobacter luteus (ALU) is a species of gram-positive bacteria in the genus Arthrobacter. A. luteus is facultatively anaerobic, pleomorphic, branching, non-motile, non-sporulating, non-acid-fast, catalase-positive, and rod-shaped.

<i>Eco</i>RI Restriction enzyme

EcoRI is a restriction endonuclease enzyme isolated from species E. coli. It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. The Eco part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain.

References

  1. ↑ "PstI (10 U/L) - Thermo Fisher Scientific". www.thermofisher.com. Retrieved 2016-04-29.
  2. ↑ 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. doi: 10.1016/S0021-9258(17)42896-1 . ISSN   0021-9258. PMID   6330092.
  3. ↑ 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. Bibcode:1981PNAS...78.1503W. doi: 10.1073/pnas.78.3.1503 . ISSN   0027-8424. PMC   319159 . PMID   6262807.
  4. ↑ 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. doi: 10.1016/S0021-9258(17)42896-1 . ISSN   0021-9258. PMID   6330092.
  5. ↑ 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. doi: 10.1016/S0021-9258(17)42896-1 . ISSN   0021-9258. PMID   6330092.
  6. ↑ 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. doi:10.1093/nar/20.24.6517. ISSN   0305-1048. PMC   334566 . PMID   1480472.
  7. ↑ Armstrong, Karen (1982). "Preferential site-dependent cleavage by restriction endonuclease PstI". Nucleic Acids Research. 10 (3): 993–1007. doi:10.1093/nar/10.3.993. PMC   326216 . PMID   6278444.
  8. ↑ Bolivar, F; Rodriguez, RL; Greene, PJ; Betlach, MC; Heyneker, HL; Boyer, HW; Crosa, JH; Falkow, S (1977). "Construction and characterization of new cloning vehicles. II. A multipurpose cloning system". Gene. 2 (2): 95–113. doi:10.1016/0378-1119(77)90000-2. PMID   344137.
  9. ↑ "PstI".
  10. ↑ Carter, Jacqueline (1980). "A Comparison of DNA Cleavage By the Restriction Enzymes SalPI and PstI". Nucleic Acids Research. 8 (21): 4943–54. doi:10.1093/nar/8.21.4943. PMC   324271 . PMID   6255438.
  11. ↑ Thomas, M (1975). "Studies on the cleavage of bacteriophage lambda DNA with EcoRI Restriction endonuclease". Journal of Molecular Biology. 91 (3): 315–328. doi:10.1016/0022-2836(75)90383-6. PMID   1102702.
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