Certain restriction endonucleases show preferential cleavage of some sites in the same substrate. In 1975, Thomas and Davis (1) observed that EcoRI (NEB #R0101) cleaves the five sites on lambda (λ) DNA nonrandomly. The site nearest the right terminus is cleaved 10 times faster than the sites in the middle of the molecule. Forsblum et al. (2) reported that EcoRI (NEB #R0101) cleaves sites on adenovirus-2 DNA at different rates. Nath and Azzolina (3) reported 10-fold and 14-fold differences among rates of cleavage of EcoRI (NEB #R0101) and HindIII (NEB #R0104) sites respectively on lambda DNA. Brown and Smith found that HgaI (NEB #R0154) cleaves certain sites on phiX174 DNA more quickly than others (4). Gingeras and Brooks (5) reported that a CTCGAG site in adenovirus-2 DNA is completely impervious to cleavage by PaeR7I (NEB #R0177), yet is readily cleaved by XhoI (NEB #R0146), an isoschizomer of PaeR7I (NEB #R0177). In this particular case, the resistance to cleavage is attributed to the surrounding sequence, a CT dinucleotide bordering the 5´ end of the site CTCGAG. Methylation of the recognition site does not appear to be the cause of the differential cleavage rates in any of the above cases.
Some restriction endonucleases require two copies of their recognition sequence before they can cleave DNA; this can lead to unusual cleavage properties. For instance, BspMI (NEB #R0502) (6), SfiI (NEB #R0123) (7) and NgoMIV (NEB #R0564) (8) are all homotetrameric proteins that bind two recognition sequences and cleave four phosphodiester bonds concurrently. These enzymes require two recognition sites in cis for optimal activity; consequently, they do not cut a molecule containing a single site easily. The requirement for two sites seems to be particularly common, although not universal, among Type IIS enzymes, which are generally monomers that transiently associate to form dimers to cleave both strands (9-11). These include such enzymes as FokI (NEB #R0109), BsgI (NEB #R0559), BpmI (NEB #R0565) and MboII (NEB #R0148) (10). Yet others such as EcoRII and NaeI (NEB #R0190) interact with two copies of their recognition sequence, one being the target for cleavage, the other serving as an allosteric effector (12-15). For these enzymes, the second, non-cleavable site can be provided either on the same DNA molecule or in the form of an activator oligonucleotide (12,13). Slow and/or resistant recognition sites on certain substrates have also been observed for HpaII (NEB #R0171), NarI (NEB #R0191) and SacII (NEB #R0157) however, significantly less is known about the mode of action of these enzymes (14).
These properties of restriction endonucleases can result in dramatic site preference. For example, plasmids containing a single site for NaeI (NEB #R0190), NarI (NEB #R0191) and BspMI (NEB #R0502) are cleaved very poorly. However, pBR322 contains four NarI (NEB #R0191)recognition sequences, and one unit of NarI (NEB #R0191) (as defined on adenovirus-2 DNA) will cut two of these sites to completion in an hour under standard conditions. An additional 50 units of enzyme will not cleave the remaining two sites to completion, even after incubation for 16 hours. Of the four NaeI (NEB #R0190) sites in pBR322, two are readily cleaved, one is cleaved moderately slow, and the fourth is cleaved 50-fold more slowly. NarI (NEB #R0191) and NaeI (NEB #R0190) each have one recognition site on λ DNA, but only partial digestion of λ can be achieved by many-fold overdigestion with either of these enzymes. SacII (NEB #R0157) has four sites on λ DNA. Three of the sites are clustered near the center of the DNA molecule. These sites are cleaved 50-fold faster than the remaining site near the right terminus (nucleotide coordinate 40,386).
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