Bacterial restriction/modification systems protect against invaders
The discovery of restriction enzymes, or restriction endonucleases (REs), was pivotal to the development of molecular cloning. REs occur naturally in bacteria, where they specifically recognize short stretches of nucleotides in DNA and catalyze double-strand breaks at or near
the recognition site (also known as a restriction site). To date, thousands of REs with distinct specificities have been described. You might wonder why bacteria harbor these potentially destructive enzymes. REs are part of a bacterial defense system against foreign DNA, such as an infectious bacteriophage. The RE sites in the bacterium’s own DNA are protected from cleavage because they have been modified by a methyltransferase that specifically modifies the RE sites. The combined activities of the endonuclease and methyltransferase are referred to as a restriction/ modification system. Today, most commercially available REs are not purified from their natural sources.. Instead, REs are usually isolated from bacteria that overexpress large quantities of REs from plasmids. These recombinant REs have often been engineered by molecular biologists to include amino acid changes that increase the catalytic activity or stability of the RE.
To understand how REs work, we will use EcoRI, one of the best-studied REs, as an example. Although the names of individual REs may sound a bit like baby talk, the nomenclature is actually very systematic and is based on its biological source. EcoRI is found naturally in the RY13 strain of Escherichia coli. Its name begins with the genus and species (Eco for E. coli), followed by a strain identifier (R for RY13), and ends with a Roman numeral that distinguishes the different REs found in the strain. Strain RY13 of E. coli contains multiple REs, but only EcoRI and EcoRV, are widely used in molecular biology.
Restriction enzymes cleave specific sites in DNA
Restriction enzymes like EcoRI are frequently called 6-cutters, because they recognize a 6-nucleotide sequence. Assuming a random distribution of A, C, G and Ts in DNA, probability predicts that a recognition site for a 6-cutter should occur about once for every 4096 bp (46) in DNA. Of course, the distribution of nucleotides in DNA is not random, so the actual sizes of DNA fragments produced by EcoRI range from hundreds to many thousands of base pairs, but the mean size is close to 4000 bp. DNA fragments of this length are useful in the lab, since they long enough to contain the coding sequence for proteins and are well-resolved on agarose gels.
EcoRI recognizes the sequence G A A T T C in double stranded DNA. This recognition sequence is a palindrome with a two-fold axis of symmetry, because reading from 5’ to 3’ on either strand of the helix gives the same sequence. The palindromic nature of the restriction site is more obvious in the figure below. The dot in the center of the restriction site denotes the axis of symmetry. EcoRI catalyzes the hydrolysis of the phosphodiester bonds between G and A on both DNA strands. The restriction fragments generated in the reaction have short single-stranded tails at the 5’-ends. These ends are often referred to as “sticky ends,” because of their ability to form hydrogen bonds with complementary DNA sequences.
REs are sometimes referred to as molecular scissors because of their ability to generate restriction fragments that terminate with defined sequences. These “sticky ends” are important for recombinant DNA technology, because they enables researchers to construct designer DNA molecules. Any two DNA molecules with compatible sticky ends can be joined together by DNA ligases that serve as the “paste” by resealing broken phosphodiester bonds. We will not be generating recombinant molecules in this class, but it is important to understand their importance to modern biology. Consider the pBG1805 and pYES2.1 plasmids. From the plasmid maps in Chapter 10, you can see that these complex plasmids were constructed by stitching together DNA sequences from evolutionary distinct sources.