The Oncologist, Vol. 7, No. 1, 82-83,
February 2002
© 2002 AlphaMed Press
FUNDAMENTALS OF CANCER MEDICINE |
The Molecular Perspective: Restriction Endonucleases
David S. Goodsell
Correspondence:
David S. Goodsell, Ph.D., Associate Professor, The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 858-784-2839; Fax: 858-784-2860; e-mail: goodsell{at}scripps.edu Website:http://www.scripps.edu/pub/goodsell
We live in a remarkable age. Physicians now have an unprecedented range of options for helping their patients. Surgery and radiology are using advanced technologies to pinpoint treatments. Chemotherapeutic approaches are showing ever-greater effectiveness and specificity, approaching the goal of a "magic bullet." And we are now entering a time when we can make changes at the most basic level, introducing changes directly into the genetic code. Gene therapy, the ability to introduce new genes into living cells, holds great promise for the treatment of many diseases, including cancer.
Restriction endonucleases are the molecules that spawned the field of biotechnology, the first of the many molecular tools that make gene therapy a reality. Bacteria build restriction endonucleases to protect themselves from attack by viruses. These enzymes cleave DNA at a specific sequence of nucleotides: for instance, one enzyme from Escherichia coli cuts the sequence GAATTC and another enzyme from Bacillus amyloliquefaciens will not cut it, but instead cuts the similar sequence GGATCC. Each species of bacterium protects its own DNA at its personal sequence by adding a bulky methyl group on a few of the bases, so only invading viral DNA, which does not have the protective methyl groups, is chopped up and destroyed. There are hundreds of forms of these restriction enzymes, made by different bacteria to cut DNA at different sequences.
Although this may seem merely like a scientific curiosity worthy of a few lines in a textbook, restriction endonucleases are almost unbelievably useful. We could not have designed a more perfect molecule-sized tool to manipulate DNA. They are custom molecular scissors, allowing us to cut DNA into well-defined, perfectly specified pieces. Hundreds of different restriction endonucleases are available commercially: you merely pick the sequence that you want cleaved and then go to the catalog and find the enzyme from the proper species of bacteria.
With these tiny molecular tools, perfected by evolution, we are able to reassemble DNA, cutting out genes of interest and inserting them in new places. Gene therapy is one of the most recent fruits of this technology. In gene therapy, a gene is spliced into a vector, which delivers the gene into living cells. Gene therapy for cancer carries a dual challenge: designing a vector that efficiently targets cancer cells and designing a gene that consistently cures or kills the transformed cell.
The vector may be a retrovirus, which has the ability to insert the gene into the cellular genome, potentially making a permanent change. In cancer treatment, this approach is taken when attempting to cure cancer cells, perhaps by replacing a faulty p53 gene. Alternatively, the gene may be carried into the cell by adenovirus. The gene is not incorporated into the cellular genome, but as the virus reproduces in the infected cell, the gene is expressed at high levels. This approach is being used to fight cancer by designing viruses that seek out cancer cells and introducing genes that kill the cell. These genes might encode a toxic protein that kills the cell directly, or a protein that makes the cell more visible to the immune system.
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Figure 1. Restriction endonucleases. Restriction endonucleases are small and stable, built to perform their job with efficiency. Most restriction endonucleases are dimeric enzymes: they wrap around the DNA, and one subunit cleaves one strand and the other subunit cleaves the complementary strand. The example shown here is EcoRI, an enzyme from Escherichia coli. The two protein subunits are colored red and orange, and the DNA strands are in blue and green. Atomic coordinates were obtained from entry 1eri at the Protein Data Bank (http://www.pdb.org).
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Figure 2. Sticky ends. A serendipitous property of many of these restriction enzymes makes the cutting and pasting of DNA particularly easy. Often the cuts on the two strands are staggered, forming overhanging single-stranded ends a few nucleotides long. These ends are termed "sticky ends" because the free bases can still form normal base pairs, gluing the two pieces back together if the conditions are mild enough. This is perfect for genetic engineering, allowing researchers to cut out pieces and reassemble them in different orders, matching pieces up when the sticky ends match.
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ADDITIONAL READING
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 Additional Reading
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Vile RG, Russell SJ, Lemione NR. Cancer gene therapy: hardlessons and new courses. Gene Ther 2000;7:2–8.[CrossRef][Medline]
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Winkler FK. Structure and function of restriction endonucleases. Curr Opin Struct Biol 1992;2:93–99.
