Restriction endonuclease endonuclease

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

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). 

Table 11.5 lists many of the commonly used restriction endonucleases and their recognition sites. Because these sites all have twofold symmetry, only the sequence on one strand needs to be designated. 

Treatment widi restriction endonuclease Agave 2 fragments, one 7 kb in size and one 3 kb in size, as judged by gel electrophoresis. 

FIGURE 11.33 Restricdon mapping of a DNA molecule as determined by an analysis of the electrophoretic pattern obtained for different restriction endonuclease digests. (Keep in mind that a dsDNA molecule has a unique nucleotide sequence and therefore a definite polarity thus, fragments from one end are distinctly different from fragments derived from the other end.)... 

FIGURE 13.3 Restriction endonuclease Ec691 cleaves double-stranded DNA. The recognition site for is the hexameric sequence GAATTC ... 

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. 

I been established to serve as a registry of convicted offenders. When a DNA sample is obtained from a crime scene, the sample is subjected to cleavage with restriction endonucleases to cut out fragments containing the STR loci, the fragments are amplified using the polymerase chain reaction, and the sequences of the fragments are determined. 

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. 

Blasiak J, Kowalik J. 1998. Interaction between organophosphoms compounds and DNA assayed by the restriction endonuclease EcoRI. Acta Univ Lodz Folia Biochim Biophys 13 31-67. 

Examples (a) nucleosome K Huger, AW Mader, RK Richmond, DF Sargent, TJ Richmond. Nature 389 251-260, 1997 (b) DNA polymerases CA Brautigam, TA Steitz. Curr. Opin. Struct. Biol. 8 54-63, 1998 (c) single-stranded binding protein Y Shamoo, AM Friedman, MR Parsons, WH Konigsberg, TA Steitz. Nature 376 362-366, 1995 (d) restriction endonucleases RA Kovall, BW Matthews. Curr. Opin. Chem. Biol. 3 578-583, 1999 (e) DNA lig-ase S Shuman. Structure 4 653-656, 1996 (f) DNA helicases MC Hall, SW Matson. Mol. Microbiol. 34 867-877, 1999 (g) zinc-finger proteins Y Choo, JW Schwabe. Nat. Struct. Biol. 5 253-255, 1998. 

The detection of restriction fi agment length polymorphisms (RFLPs) facilitates prenatal detection of hereditary disorders such as sickle cell trait, beta-thalassemia, infant phenylketonuria, and Huntington s disease. Detection of RFLPs involves cleavage of double-stranded DNA by restriction endonucleases, which can detect subtle alterations in DNA that affect their recognized sites. Chapter 40 provides further details concerning the use of PCR and restriction enzymes for diagnosis. 

Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms. 

Table 40-2. Selected restriction endonucleases and their sequence specificities ...

---