Smith & Wilcox discovered in 1969 that bacteria use enzymes to cut DNA at specific sequences, protecting their own DNA through methylation. Using radioactive labeling and sedimentation, they mapped a six-base recognition site and showed the enzyme makes clean double-strand breaks. This discovery of restriction endonucleases became foundational to genetic engineering.
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Thank you for watching Transformative Advances in Molecular Biology, a retrospective look at critical events in the history of the discipline. The presentations in this series were prepared by graduate students in a journal colloquium at the University of Florida, supervised by Mark Settles and Kevin Folta.
Hi, this is Greg Maloney and Haiyan Jia, and we will be talking about the discovery of restriction enzymes. 2 groups first studied these endonucleases. They were Smith and Wilcox and Kelly and Smith. Authors Smith and Wilcox in 1969 were studying phages in Haemophilus influenzae. They knew that the phage 22 is deactivated in this bacterium and cannot reproduce. This phenomenon is where the term restriction came from, because the virus was restricted from growth in certain host species. One of the first experiments they performed was to incubate H. influenzae DNA And phage DNA with the cellular extract. This graph has time on the x-axis and viscosity on the y-axis, and it shows that the viscosity of both samples were measured, and it was found that the phage DNA became much less viscous while the bacterial DNA did not lose viscosity. The degradation of phage DNA showed that some factor contained within the bacterial extract could selectively restrict To further investigate this mysterious phage degradation, the researchers made p32-labeled phage DNA. They also purified the cell fraction with the enzymatic restriction property and incubated it with the labeled phage DNA. TCA is a chemical that precipitates large pieces of DNA and solubilizes very small pieces. TCA is a very strong acid. They used TCA in addition with the sucrose sedimentation on double-stranded and single-stranded DNA and found that only double-stranded DNA was digested by the enzymatic fraction, which was a clue for the mechanism of action. The figure on the left here shows the sucrose sedimentation plots of phage and bacterial DNA after digestion. The phage DNA appearing in a higher fraction because of its small pieces. The figure on the right, from top to bottom, shows single-stranded DNA without enzyme, single-stranded DNA with enzyme, and double-stranded DNA with enzyme in a sucrose gradient. The researchers then wanted to find the details of the cleavage reaction with a series of reactions with phage DNA. They first digested with the endonuclease fraction. Then they treated with a phosphatase to release all 5' nucleotide ends, after which they treated with kinase to add P-32 to all the free 5' ends. They recorded counts of P-32 label on the treated DNA and found that about 32 times more label than was on undigested DNA. From this, and knowing the structure of DNA, they could conclude that the enzyme created a 3' hydroxyl 5' phosphoryl cleavage.
In order to identify the base sequence of recognition site, in Kelly and Smith paper, add labeling techniques were used. This scheme shows the general method for analysis of 5-terminal nucleotide sequences. First, uniformly P-33 labeled phage T7 DNA was digested to a limit product with endonuclease Arc. Then the 5' phosphoryl ends were removed with alkali phosphatase. P-32-labeled phosphoryl groups were then transferred from ATP to the 5' termini using polynucleotide kinase. Finally, 5' terminal mono-, di-, and trinucleotides were isolated after digestion with various nucleases. First, 5' terminal mononucleotides were analyzed. Terminally labeled DNA was hydrolyzed to mononucleotides by By sequential digestion with DNase and phosphodiesterase, then analyzed by electrophoresis. Figure 2 shows dositometer tracing of unscreened A and screened B autoradiographs. It revealed that AMP and GMP were the only mononucleotides which contained P32. The conclusion is, endonuclease R cleaves both strands of DNA duplex on the 5' side of a purine nucleotide.
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Then, 5-terminal dinucleotides were analyzed. Terminally labeled DNA was digested sequentially with DNase and exonuclease I and fractionated by electrophoresis. Figure 3 shows 2 distinct p32-containing dinucleotide species, 2A and 2B. Upon digestion to mononucleotides, the radioactivity associated with Only AMP and GMP, as showed by Table 2, indicating species 2A is AA, species 2B is GA. The conclusion is endonuclease R cleaves at purine A. 5-terminal trinucleotides were analyzed. Terminally labeled DNA was digested sequentially with DNase and phosphodiesterase. Trinucleotide fraction was analyzed by electrophoresis. Figure 5 shows there are 2 p32-containing trinucleotide species, 3A and 3B. After separation of 3A and 3B from contaminating trinucleotide by electrophoresis, as showed by Figures 6 and 7, species 3A was identified as AAC, species 3B was GAC. The conclusion is endonuclease R cleaves at purine AC.
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They also identified the nucleotide sequence at 3 ends. The scheme on top left shows possible structures for substrate region. Structure A, the nicks are exactly opposite one another, so the 3 ends of the fragments produced are complementary to the 5 ends. B and C, the nicks are staggered. So the 3' ends of the fragments produced are not necessarily complementary to the 5' ends. In Figure 9, uniform AP32-labeled endonuclease R limit product was digested with micrococcal nuclease, and the 3' terminal dinucleoside monophosphates were analyzed by electrophoresis. 2 major radioactive species were observed, and each species were hydrolyzed with different phosphodiesterase. As showed by Figure 10, indicating species 1 was TC, species 2 was TT. This was the expected result for an even break. Finally, they analyzed the size of the recognition sequences. Endonuclease R-limited product labeled at 5' with P32 was digested with DNase. The Tetranucleotide was fractionated by electrophoresis. 6 distinct T32-containing tetranucleotides were observed, indicating that the 4th nucleotide from the 5' end of the LEMIT product is not spencified. The conclusion is, the recognition region is 6 nucleotide pairs.
In summary, the researchers found that endonuclease R produces a 5' phosphoryl hydroxyl double-strand break in foreign DNA, but not in host DNA, which may be protected by methylation or some other unknown modification. They also found that in phage DNA, these breaks occur about every 1,000 bases, which gives them some idea of the length of the recognition sequence. They found that endonuclease R recognizes the nucleotide sequence guanine, thymine, pyrimidine, purine, adenine, and cytosine. This endonuclease R is today known as HindII, which is a Type II restriction enzyme. And altogether, these reports made very significant changes to how recombinant DNA technology is studied today and will be in the future.
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