Methods in Molecular Biology • 16 Enzymes of Molecular Biology

Restriction Enzymes 185

removal of
single
restriction mis-match
site I primer
aa change..

change of

restriction

site 2

1. Hybridization:
   ss vector DNA +
   ds gap fragment +
   mis-match primer


2. FiIHn with DNA polymerase
   and ligase


3. Transformation into a repair
   deficient strain


4. Cleavage of the plasmid DNA
   with restriction enzyme 1
   (enrichment of mutant plasmid)


5. Transformation into a normal
   strain


6. Screening with restriction
   enzyme 2 (identification of
   mutant plasmid)


7. DNA sequencing

Fig. 3. Use of restriction enzyme catalyzed DNA cleavage to increase the marker

yield of a site-directed mutagenesis experiment and to screen for positive clones.

desired mutant. Identification of positive clones can be done by screen-

ing for a restriction site introduced together with the desired mutation

on the mismatch primer or direct sequencing (Fig. 3).

Note Added in Proof

After completion of this review, many new discoveries regarding

restriction enzymes were made, only a few of which and only those

that are of general importance are considered in this note.

The structure of the EcoRI-DNA recognition complex has been

revised (199): According to the new structure (reviewed in ref. 200),

the recognition interactions comprise 16 protein-base hydrogen bonds

to the purines and pyrimidines, and also van der Waal's contacts to all

of the pyrimidines of the recognition sequence. Furthermore, hydro-

gen bonds and electrostatic contacts to the phosphates within and

outside of the recognition sequence most likely contribute to the speci-

ficity of DNA binding by EcoRI. Direct interactions are buttressed by

indirect ones between amino acid residues involved directly in base-

specific contacts and amino acids nearby. In contrast to the earlier