Endonucleases, Restriction occurence

Nebularine. Nebularine(44) is a naturaHy occurring purine riboside isolated from S.jokosukanensis (1,3,4). It is phosphorylated, and inhibits purine biosynthesis and RNA synthesis, but is not incorporated into RNA by E. coli RNA polymerase. It has also found appHcation as a transition state analogue for treatment of schistosomiasis and as a substrate for the restriction endonuclease, Hindll (138—141). 

The first step in DNA sequencing is to cleave the enormous chain at known points to produce smaller, more manageable pieces, a task accomplished by the use of restriction endonucleases. Each different restriction enzyme, of which more than 3500 are known and approximately 200 are commercially available, cleaves a DNA molecule at a point in the chain where a specific base sequence occurs. For example, the restriction enzyme Alul cleaves between G and C in the four-base sequence AG-CT. Note that the sequence is a palindrome, meaning that the sequence (5 )-AGCT-(3 ) is the same as its complement (3 )-TCGA-(5 ) when both are read in the same 5 — 3 direction. The same is true for other restriction endonucleases. 

Restriction endonuclease (Section 28.6) An enzyme that is able to cleave a DNA molecule at points in the chain where a specific base sequence occurs. 

To handle the mass of existing data, powerful computer programs have been developed and various graphical procedures have also been developed to help the human mind comprehend the results.654 655 One important problem is to define and locate what are called consensus sequences. The problem is best illustrated by examples.654 The cleavage site for the EcoRI restriction endonuclease is GAATTC. There is no ambiguity. In a DNA of random sequence this would be expected to occur by chance in about (1 / 4)6 nucleotides (4 kb). On the other hand, the Hmll restriction endonuclease cleaves within the consensus sequence GTYRAC where Y = C or T and R = A or G. 

Restriction endonuclease EcoRI is able to cut a chain in dsDNA which has a chiral phophorothioate group at the specific cleavage site.838 Tire reaction occurs with inversion of configuration at phosphorus, suggesting direct in-line attack by a hydroxyl ion generated from H20. 

Interestingly, transposon Tn7 behaves in a similar way but, in this case, the 5 endonuclease activity is supplied by a separate enzyme whose structure resembles that of a type II restriction enzyme (30), and cleavage occurs cleanly at the transposon tip rather than within the TE. Transposition of Tn7, like most bacterial elements, does not leave a scar. In both the Tc-Mariner transposon group and the Tn7 family of transposons, the transposase then cleaves and transfers the 3 end in a true DDE transposition reaction (Fig. le). 

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