End of the “restriction enzyme era” in molecular biology?
09.15.08 by Syam Anand
Restriction enzymes are the workhorses of modern recombinant DNA laboratories. They cut DNA at specified positions by recognizing specific sequences on DNA (such as the order of G, A, T and C).
http://www.youtube.com/watch?v=-sI5vy-cD2g
Presently, the methods to engineer recombinant DNA molecules with predictable coding specificities rely mostly on the ability of these enzymes to cut DNA into predictable fragments, which can be subsequently ‘pasted’ in specific ways using DNA ligases thus generating new coding information. In the classic work that first described the generation of recombinant DNA molecules in E. coli1 and since then, the term recombinant DNA meant mostly the generation and use of mostly artificial combinations of DNA fragments through the use of restriction enzymes. In nature, the ‘real’ DNA recombination reaction drives evolution in incremental steps and it’s products are subjected to selection pressure due to its tie up with cell viability. Until recently and even as we write, molecular biologists the world over, are stuck with restriction enzymes for generating recombinant DNA molecules.
The current spectrum of our understanding of basic cellular processes such as how the genetic material is replicated and segregated into daughter cells, how damaged DNA is recognized and repaired by the cellular machinery devoted for this cause, how intracellular localization and trafficking of molecules take place and how populations of different kinds of cells (such as in the immune system or during the spread of cancer cells during metastasis) localize to various parts of the body by whole body imaging, to name just a few, have been driven to a large extent by our ability to interrogate these processes in creative ways with the help of mutational DNA libraries and gene fusions of various kinds. The economic generation of recombinant DNA molecules to answer complex biological questions is however limited by our ability to identify, characterize and commercialize restriction enzymes with novel specificities, even though the currently available enzymes has allowed us to do wonders. Manipulating DNA molecules that had no cut sites for user-friendly restriction enzymes, large genomic fragments where the enzyme specificities are represented multiple times or where the sites are not at desirable positions seemed to be nearly insurmountable barriers for path-breaking discoveries. It appears that DNA recombination can overcome these barriers in order to generate recombinant DNA molecules in a practical and user-friendly way.
One should mention here that recombination-driven generation of DNA clones would not come as a surprise for yeast geneticists. They have been continuously tapping the power of in vivo recombination to generate recombinant clones in vivo. Yeast cells generate recombinant DNA by Rad-51 mediated homologous recombination with high efficiency. All one needed was to simply transform yeast cells harboring the target plasmid with a dsDNA containing necessary length of homology at the ends and the desired sequence changes and wait for in vivo recombination to take over2. Biologists have also extensively used recombination-dependent cloning by exploiting the Cre-lox recombination reaction, which also did not involve the use of restriction enzymes or ligases3. However, the requirement of particular DNA sequences for the Cre-lox system (known as att sites) continued to act as a barrier for routine cloning experiments. Various groups have also utilized the lambda Red/ET recombination to create recombinant molecules in vivo. Thus, any DNA molecule with homologous ends (similar sequences) can be precisely joined together in certain strains of E. coli cells that express phage-derived protein pairs, either RecE/RecT from the Rac prophage, or Red?/Red? from ? phage4,5.
The recent discovery of a viral DNA polymerase that can pair DNA segments containing sequence homology (same DNA sequence) at their ends seems to be a revolution-in-the-making as far as the ability to generate recombinant DNA molecules in a tube goes. The underlying reactions that ‘paste’ the DNA fragments together do not employ classical recombination reactions that involve strand-exchange and DNA synthesis. However, it is more similar to recombination reactions than restriction enzyme/ligase-mediated cutting and pasting of DNA fragments. A single enzyme6 performs the reactions that search for homology at the ends of DNA and then join them together in a matter of few minutes. Fragments containing homologous sequences at the ends can be generated either by restriction enzyme digestion or PCR and pasted together in a matter of few minutes. Especially attractive is it usefulness for large-throughput screens where DNA fragments have to be shuttled between different DNA vectors. In future, it is possible that more efficient enzymes are discovered that carry out this reaction more efficiently. If the research community adapts to the new technology, this could be signal coming of the end of a grand era in recombinant DNA technology that was dominated by restriction enzymes.
One can envision large freezers stuffed with restriction enzymes of various specificities on the inside and buffer and compatibility charts on the outside, becoming a thing of past (just like the meter long sequencing gel apparatus, which appear now as if they are from the stone-age). Restriction enzymes however, would still have a place in biology and in our hearts and they will continue to do what nature intended them to do- protect the genomes of prokaryotes.
References:
1. Cohen, S. N., Chang, A. C. Y., Boyer, H. W. and Helling, R. B. Construction of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Sciences of the United States of America, 1973, 70: 3240-3244.
2. Wendland, J. PCR-based methods facilitate targeted gene manipulations and cloning procedures. Current Genetics, 2003, 44: 115-123.
3. Liu, Q., Li, M. Z., Leibham, D., Cortez, D. and Elledge, S. J. The uni-vector system, a method for rapid construction of recombinant DNA without restriction enzymes. Current Biology, 1998, 8: 1300-1309.
4. Muyrers, J. P. P, Zhang, Y and Stewart, A. F. Techniques: Recombinogenic engineering –new options for cloning and manipulating DNA. Trends in Biochemical Sciences, 2001, 5: 325-31.
5. Copeland, N.G, Jenkins, N.A. and Court, D. L. Recombineering: a powerful new tool for mouse functional genomics. Nature Reviews Genetics, 2001, 10: 769-779.
6. Hamilton, M. D., Nuara, A. A., Gammon, D. B., Buller, R. M., and Evans, D. H. Duplex strand joining reactions catalyzed by vaccinia DNA polymerase. Nucleic Acids Research, 2007, 35:143-151.
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