Cutting of DNA by Restriction Enzymes

It might seem logical for the DNA to be cut at the recognition site where the restriction enzyme binds. This is often true, but not always. There are two major classes of restriction enzyme that differ in where they cut the DNA, relative to the recognition site.

Type I restriction enzymes cut the DNA a thousand or more base pairs away from the recognition site. This is done by looping the DNA around so that the enzyme binds both at the recognition site and the cutting site (Fig. 22.02). Since the exact length of the loop is not constant, and since the base sequence at the cut site is not fixed, these enzymes are of little practical use to molecular biologists. Even more bizarre is that type I restriction enzymes are suicidal. Most enzymes carry out the same reaction over and over again on a continual stream of target molecules. Each molecule of a type I restriction enzyme can cut DNA only a single time and then it is inactivated!

Type I restriction systems consist of a single protein with three different subunits. One subunit recognizes the DNA, another methylates the recognition sequence and the third cuts the DNA at a distance from the recognition sequence. In type II restric-

isoschizomers Restriction enzymes from different species that share the same recognition sequence type I restriction enzyme Type of restriction enzyme that cuts the DNA a thousand or more base pairs away from the recognition site

Different restriction enzymes may share the same recognition sequence although they do not necessarily cut at precisely the same place.

Type I restriction enzymes cut the DNA a long way from the recognition sequence.

FIGURE 22.02 Type I Restriction Enzyme

Type I restriction enzymes have three different subunits. The specificity subunit recognizes a specific sequence in the DNA molecule. The modification subunit adds a methyl group to the recognition site. If the DNA is non-methylated, the restriction subunit cuts the DNA, but at a different site, usually over 1000 base pairs away. In the EcoK restriction enzyme, the subunits are HsdS, HsdM, and HsdR.

subunit

HsdM

subunit

Modification subunit

HsdM

HsdR

Restriction subunit

HsdR

Type I Restriction

Enzyme (eg. EcoK)

Binding

FIGURE 22.02 Type I Restriction Enzyme

Type I restriction enzymes have three different subunits. The specificity subunit recognizes a specific sequence in the DNA molecule. The modification subunit adds a methyl group to the recognition site. If the DNA is non-methylated, the restriction subunit cuts the DNA, but at a different site, usually over 1000 base pairs away. In the EcoK restriction enzyme, the subunits are HsdS, HsdM, and HsdR.

Cutting

Type II restriction enzymes cut the DNA with in the recognition sequence. Some generate blunt ends, others give sticky ends.

Sticky ends are more convenient than blunt ends when joining together fragments of DNA using DNA ligase.

tion systems the restriction endonuclease and the methylase are two separate proteins that operate independently but recognize the same DNA sequence.

Type II restriction enzymes cut the DNA in the middle of the recognition site. Since the exact position of the cut is known, these are the restriction enzymes that are normally used in genetic engineering. There are two different ways of cutting the recognition site in half. One way is to cut both strands of the double stranded DNA at the same point. This leaves blunt ends as shown in Figure 22.03. The alternative is to cut the two strands in different places, which generates overhanging ends. The ends made by such a staggered cut will base pair with each other and consequently they are known as sticky ends.

Enzymes that generate sticky ends are the most useful. If two different pieces of DNA are cut with the same restriction enzyme or enzymes that generate the same overhang, the same sticky ends are generated. This allows fragments of DNA from two different original DNA molecules to be bound together by matching the sticky ends (Fig. 22.04). Such pairing is temporary since the pieces of DNA are only held together by hydrogen bonding between the base pairs, not by permanent covalent bonds. Nonetheless, this assists the permanent bonding of the sugar-phosphate backbone by DNA ligase. When two sticky ends made by the same enzyme are ligated, the junction may be cut apart later by using the same enzyme again. However, if two sticky ends made by two different enzymes are ligated together, a hybrid site is formed that cannot be cut by either enzyme (as would happen with BamHI and BglII in Fig. 22.04).

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