Restriction Enzymes - Background
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Restriction Enzymes

Background Paper

Access Excellence Classic Collection


Introduction

Watson and Crick's description, in 1953, of the double helical structure of the DNA molecule (See Classic Collection- The Structure of DNA) opened the door to a new era in biological understanding and research. Scientists, now knowing the molecular structure of the hereditary molecule, could begin both to elucidate and to manipulate its function. These new studies were, however, dependent on the discovery and use of the many enzymes that are able to modify or join existing DNA molecules, or to aid in the synthesis of new DNA molecules.

DNA enzymology

The late 1950's and the decade of the 60's saw enormous breakthroughs in DNA enzymology. For example, it was during this time that Arthur Kornberg and colleagues isolated DNA polymerase (1955), B. Weiss and C.C. Richardson isolated DNA ligase (1966), and H.O. Smith, K.W. Wilcox, and T.J. Kelley isolated and characterized the first sequence specific restriction nuclease (1968). These enzymes, respectively, play roles in the synthesis of DNA molecules, the attachment of two or more DNA molecules to one another, and the breaking of DNA molecules into fragments. Importantly, these enzymes make it possible to create entirely new kinds of DNA molecules and, equally important, to manipulate the functioning of the genes located on these new molecules.

Phage growth restriction

It had been known since the 1950's that phage particles that grow well and efficiently infect one strain of bacteria are often unable to grow well and infect other strains of the same bacterial species. In addition, phage particles that do succeed in infecting a second strain often show the opposite pattern: they are able to efficiently infect the second strain while growing only poorly in the original strain. A series of studies showed that phage particles that efficiently grow and infect host cells have DNA molecules that have been chemically modified by the addition of methyl groups to some of their adenine and/or cytosine bases, while the DNA of poorly infecting phage particles does not show this pattern of chemical modification or "methylation." Phage particles with unmethylated DNA do not grow and infect efficiently because their DNA molecules are cleaved and degraded by enzymes of the host cell, while methylated DNA is protected from this degradation. This phenomenon of degrading unmethylated DNA destroys the growth ability of the phage, and is responsible for the pattern of growth restriction described above.

Methylase and nuclease

In the late 1960's, scientists Stewart Linn and Werner Arber isolated examples of the two types of enzymes responsible for phage growth restriction in Escherichia coli (E. coli) bacteria. One of these enzymes methylated DNA, while the other cleaved unmethylated DNA at a wide variety of locations along the length of the molecule. The first type of enzyme was called a "methylase" while the other was called a "restriction nuclease." These enzymatic tools were important to scientists who were gathering the tools needed to "cut and paste" DNA molecules. What was needed now was a tool that would cut DNA at specific sites, rather than at random sites along the length of the molecule, so that scientists could cut DNA molecules in a predictable and reproducible way.

Site-specific nuclease

This important development came when H.O. Smith, K.W. Wilcox, and T.J. Kelley, working at Johns Hopkins University in 1968, isolated and characterized the first restriction nuclease whose functioning depended on a specific DNA nucleotide sequence. Working with Haemophilus influenzae bacteria, this group isolated an enzyme, called HindII, that always cut DNA molecules at a particular point within a specific sequence of six base pairs. This sequence is:

5' G T ( pyrimidine: T or C) ( purine: A or G) A C 3'
3' C A ( purine: A or G) ( pyrimidine: T or C) T G 5'

They found that the HindII enzyme always cuts directly in the center of this sequence. Wherever this particular sequence of six base pairs occurs unmodified in a DNA molecule, HindII will cleave both DNA backbones between the 3rd and 4th base pairs of the sequence. Moreover, HindII will only cleave a DNA molecule at this particular site. For this reason, this specific base sequence is known as the "recognition sequence" for HindII.

HindII is only one example of the class of enzymes known as restriction nucleases. In fact, more than 900 restriction enzymes, some sequence specific and some not, have been isolated from over 230 strains of bacteria since the initial discovery of HindII. These restriction enzymes generally have names that reflect their origin--The first letter of the name comes from the genus and the second two letters come from the species of the prokaryotic cell from which they were isolated. For example EcoRI comes from Escherichia coli RY13 bacteria, while HindII comes from Haemophilus influenzae strain Rd. Numbers following the nuclease names indicate the order in which the enzymes were isolated from single strains of bacteria. Nucleases are further described by addition of the prefix "endo" or "exo" to the name: The term "endonuclease" applies to sequence specific nucleases that break nucleic acid chains somewhere in the interior, rather than at the ends, of the molecule. Nucleases that function by removing nucleotides from the ends of the molecule are called "exonucleases."

Endonucleases and DNA fragments

A restriction endonuclease functions by "scanning" the length of a DNA molecule. Once it encounters its particular specific recognition sequence, it will bond to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones of the double helix. The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease used to cleave the molecule in the first place. Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no matter what DNA molecule it is acting on. Once the cuts have been made, the DNA molecule will break into fragments.

Endonucleases and sticky ends

Not all restriction endonucleases cut symmetrically and leave blunt ends like HindII described above. Many endonucleases cleave the DNA backbones in positions that are not directly opposite each other. For example, the nuclease EcoRI has the following recognition sequence:

5' G A A T T C 3'
3' C T T A A G 5'

When the enzyme encounters this sequence, it cleaves each backbone between the G and the closest A base residues. Once the cuts have been made, the resulting fragments are held together only by the relatively weak hydrogen bonds that hold the complementary bases to each other. The weakness of these bonds allows the DNA fragments to separate from one each other. Each resulting fragment has a protruding 5' end composed of unpaired bases. Other enzymes create cuts in the DNA backbone which result in protruding 3' ends. Protruding ends--both 3' and 5'-- are sometimes called "sticky ends" because they tend to bond with complementary sequences of bases. In other words, if an unpaired length of bases (5' A A T T 3') encounters another unpaired length with the sequence (3' T T A A5') they will bond to each other--they are "sticky" for each other. Ligase enzyme is then used to join the phosphate backbones of the two molecules. The cellular origin, or even the species origin, of the sticky ends does not affect their stickiness. Any pair of complementary sequences will tend to bond, even if one of the sequences comes from a length of human DNA, and the other comes from a length of bacterial DNA. In fact, it is this quality of stickiness that allows production of recombinant DNA molecules, molecules which are composed of DNA from different sources, and which has given birth to an industry!


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Source: Pamela Peters, Ph.D., Access Excellence, Genentech, Inc.


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