Download demonstating sequence-specific cleavage by a restriction enzyme

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Classic Experiment
9.2
DEMONSTATING SEQUENCE-SPECIFIC
CLEAVAGE BY A RESTRICTION ENZYME
acteria exhibit a phenomena, known as host restriction, whereby they can
B
both recognize and cleave foreign DNA, preventing it from interfering with
the bacterial life cycle. By purifying and characterizing one of the enzymes
involved in host restriction, Hamilton Smith gave molecular biology one of its
most important tools, an enzyme that cleaves DNA at a specific sequence.
Background
At the time of Hamilton Smith’s work, host restriction was
a well-characterized, yet highly intriguing phenomenon. It
was well known that DNA from one species of bacteria
could not be used to transform a second species of bacteria. When researchers simply mixed DNA from one bacteria with a lysate from a second bacterial species, the DNA
was cleaved. The bacteria had evolved a system to recognize and cleave foreign DNA. In 1965, Werner Arber
hypothesized that bacteria must produce an enzyme capable of recognizing and cleaving foreign DNA at specific
sequences. How did a bacterium determine which DNA
was foreign, and which was its own? It seemed unlikely
that a bacterium could exclude specific sequences in its
genome, from the action of this nuclease. More likely, a
bacterium somehow modified its own DNA at these
sequences, so it could be spared from cleavage. The existence of a second enzyme was thus hypothesized, one that
could modify the DNA by methylation at the site where
cleavage occurred, thereby preventing cleavage by the
sequence-specific nuclease.
With these hypotheses in hand, the hunt for the
enzymes could begin. In 1968, Mathew Meselson reported
the purification from E. coli of one of these enzymes now
called restriction enzymes or restriction endonucleases.
Although the E. coli enzyme catalyzed the cleavage of
non-E.coli DNA, Meselson could not demonstrate that
this cleavage was sequence specific. In fact, proving that
these bacterial enzymes cleave DNA at a specific sequence
would be a tricky manner, as this research was conducted
before the advent of the relatively simple DNA-sequencing
techniques now available. Following on Messelson’s
work, Smith set out to purify a second restriction enzyme,
this time from H. influenzae, and to demonstrate that it
does indeed cleave DNA in a sequence-specific manner.
The Experiment
The first step in the successful purification of a new
enzyme is devising an assay that measures the known
activity of the enzyme as it is being purified. The activity
of a restriction enzyme is to catalyze the cleavage of foreign DNA, so this was the logical activity to monitor. To
do so, Smith took advantage of the fact that genomic
DNA from bacteria is quite viscous, however as nucleases
begin to degrade the bacterial DNA, its the overall viscosity decreases. Therefore, Smith could monitor the purification of his restriction enzyme by measuring the decrease
in viscosity of a foreign DNA after treatment with a sample of the protein after each step in the purification
scheme. Smith mixed cell extracts of H. influenzae with
intact DNA from either H. influenzae or the Salmonella
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bacteriophage P22. Using a device called a viscometer, he
measured how the DNA from P22 became less viscous
over time, while the H. influenzae DNA displayed no
change in viscosity. This would be the assay he would use
throughout the purification scheme.
Smith used a variety of established methods to separate
bacterial lysates into smaller pools of proteins. Each
method separated the lysate based on a different physical
property of the proteins (and other biomolecules) that
make up the lysate. This allowed the lysate to be divided
into subsamples known as fractions. After each step in the
purification, every fraction was separately assayed for the
ability to cleave P22 DNA. Fractions that contained the
enzyme activity were subjected to yet another purification
method, and the process was continued until a pure
enzyme was obtained. Smith called the purified restriction
enzyme endonuclease R.
Next Smith determined some of the basic characteristics of endonuclease R. He used endonuclease R to digest
DNA from the bacteriophage T7, then estimated the number of sites where the DNA was cleaved. He discovered
that endonuclease R did not completely degrade T7 DNA,
but rather cleaved it at approximately 40 sites. Since T7
DNA contains approximately 40,000 bases, cleavage
occurred at only 0.1 percent of the possible sites. This
observation suggested to Smith that Arber’s hypothesis
was correct—the enzyme was cleaving the DNA at specific sequences. In order to prove that this was the case,
Smith had to determine the sequence at which the enzyme
cleaved the DNA, which he called the recognition site.
With the purified enzyme and evidence of sequencespecific DNA cleavage, Smith focused his attention on
determining the sequence of the recognition site. At this
time, the 1960s, the only known method of DNA sequencing was to sequentially remove nucleotides from the 5 ¿
end of DNA and determine their identity by thin layer
chromatography (TLC). Smith devised a scheme to
sequence the recognition site by using known enzymes to
cleave the ends of a DNA strand into small pieces that
could be analyzed by TLC (see Figure).
Smith began by labeling the 5 ¿ end of endonuclease Rdigested DNA with a radioactive marker, 32P. This was
accomplished by first treating the DNA with alkaline phosphatase, an enzyme that catalyzes the removal of 5 ¿ phosphate groups from polynucleotides. Next, polynucleotide
kinase, which catalyzes addition of phosphate to the 5 ¿ end
of polynucleotides, was used to transfer 32P from labeled
ATP to the terminal nucleotide. Now, the terminal
nucleotide could be easily distinguished from the rest of the
nucleotides, by virtue of its specific radioactive label. The
DNA was then digested to single nucleotides with a nuclease called pancreatic DNase. The only 32P-labeled
nucleotides observed contained adenine (A) and guanine
(G). Since no 32P-labeled nucleotide containing cytosine (C)
Recognition site
3'
5'
5'
3'
Endonuclease R
P
3'
5'
5'
3'
P
Alkaline phosphatase
5'
3'
3'
5'
Polynucleotide kinase
[32P] ATP
P*
3'
5'
5'
3'
*P
Digestion with various
nucleases
*P
Mononucleotides
n=2
P*
P*
*P
Dinucleotides
n=3
P*
*P
Trinucleotides
n=1
Schematic representation of the method used to determine the
nucleotide sequence recognized by endonuclease R. T7 bacteriophage DNA was digested with endonulcease R. After removal of
the 5’ phosphate, and addition of a 32P label, the 5’ end-labeled
DNA was digested with a variety of nucleases. 32P-labeled
mononucleotides, dinucleotides, and trinucleotides were isolated
and analyzed to determine the recognition site sequence.
[Adapted from T. J. Kelly and H. O. Smith, 1970, J. Mol.Biol.
51:393.]
or thymine (T) was detected, Smith deduced that the first
base in the recognition sequence must be a purine.
To determine the second base in the recognition site,
Smith used a nuclease that could not cleave 5 ¿ terminal dinucleotides. In other words, the entire DNA sample was
digested into single nucleotides except the final two, which
remained in dinucleotide form. Since the DNA previously
had been cleaved with endonuclease R, the 5 ¿ terminal dinulceotides are the first two bases in the recognition site.
Smith first separated the dinucleotides from the single
nucleotides. When he analyzed the dinucleotides by TLC,
he found only two species of dinucleotides that carried the
32
P label. The identity of the 32P-labeled dinucleotides was
determined by comparing their migration to that of dinucleotides of known sequence. One of the species displayed
the same migration as the dinucleotide GA; the other
migrated with the dinucleotide AA. Smith concluded that
the second base in the recognition sequence was adenine.
Analysis of the rest of the recognition site would not be
so easy, but Smith’s persistence paid off. He identified the
third base in the recognition site as cytosine using a similar,
but slightly more complicated method. He further showed
this to be the end of the recognition sequence by showing
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that the fourth nucleotide could contain any base. Now he
knew digestion of double stranded DNA with endonuclease
R creates several smaller fragments with identical 5’ ends,
which contain the sequence purine-adenine-cytosine. Since
the DNA strands are complementary, the only possible way
this could occur is if the enzyme recognized a six-base
sequence that appeared the same on either strand, known as
a pallindromic sequence. Therefore, Smith concluded that
endonuclease R recognized and cleaved DNA specifically at
the sequence GTPyPuAC.
Discussion
Although the first restriction enzyme had been purified
two years before Smith reported his work on endonuclease R, he was the first to demonstrate sequence-specific
cleavage. He then went on to purify and characterize the
methylase that allows DNA from H. influenzae to escape
cleavage. By using these sequence-specific restriction
enzymes, researchers could now cleave DNA at specific
sites. The impact of restriction enzymes on biological
research over cannot be overstated. Early on, these
enzymes were used for mapping plasmid and phage DNA.
Now they are routinely used for probing the structure of
both specific genes and of DNA from individuals. In addition, they are primary reagents in the construction of gene
expression vectors, allowing DNA from different sources
to be cleaved at specific sequences, then joined with similarly cleaved DNA. The results are seen everyday in laboratories employing recombinant DNA technologies. In
1978, Hamiliton Smith was awarded the Nobel Prize for
Physiology and Medicine in recognition of his powerful
discovery.