THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 285, No. 3, Issue of January 15, p.

e2, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Classics
A PAPER IN A SERIES REPRINTED TO CELEBRATE THE CENTENARY OF THE JBC IN 2005

JBC Centennial
1905–2005
100 Years of Biochemistry and Molecular Biology

The Characterization of Restriction Endonucleases:

the Work of Hamilton Smith
Purification of the HhaII Restriction Endonuclease from an Overproducer Esche-
richia coli Clone
(Kelly, S., Kaddurah-Daouk, R., and Smith, H. O. (1985) J. Biol. Chem. 260,
15339 –15344)
Catalytic Properties of the HhaII Restriction Endonuclease

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(Kaddurah-Daouk, R., Cho, P., and Smith, H. O. (1985) J. Biol. Chem. 260,
15345–15351)
Hamilton Othanel Smith was born in 1931
in New York City. In 1937, he and his family
moved to Champaign-Urbana, Illinois, be-
cause his father had joined the faculty of the
department of education at the University of
Illinois. As a boy, Smith was interested in
chemistry, electricity, and electronics, and
he spent many hours with his brother in
their basement laboratory, which was
stocked with supplies purchased from their
paper route earnings. Smith attended a
small college preparatory school called the
University Laboratory High School and
graduated in 3 years largely due to his sci-
ence teacher who allowed him to complete
chemistry and physics during the summer.
After finishing high school, Smith en-
Hamilton O. Smith rolled at the University of Illinois, major-
ing in mathematics. During his sophomore
year, his brother showed him a book on mathematical modeling of central nervous system
circuits by Nicolas Rashevsky. This caught his interest, and after transferring to the
University of California, Berkeley, Smith immersed himself in courses in cell physiology,
biochemistry, and biology. A guest lecture by Journal of Biological Chemistry (JBC) Classic
author George Wald (1) describing his studies of retinal biochemistry soon converted Smith
into a devoted student of visual physiology and eventually motivated him to apply to
medical school.
In 1952, Smith began his studies at the Johns Hopkins University Medical School. He
received his M.D. 4 years later and went to Barnes Hospital in St. Louis for a medical
internship. However, in 1957, Smith was called up in the Doctor Draft and joined the U.S.
Navy. He finished his Navy service in 1959 and moved to Detroit to begin a medical residency
training at the Henry Ford Hospital. There he became interested in bacteriophage and decided
that this would be the focus of his research.
So, in 1962, Smith began his research career with Myron Levine in the department of human
genetics at the University of Michigan in Ann Arbor. He and Levine carried out a series of
studies demonstrating the sequential action of the phage P22 C-genes, which controlled
e2 This paper is available on line at http://www.jbc.org
 Classics e3
lysogenization. They also discovered the gene controlling prophage attachment, now known as
the int gene, and carried out a study of defective transducing particles formed after induction
of int mutant prophage.
In 1967, Smith joined the faculty of Johns Hopkins University as an assistant professor of
microbiology and continued his bacteriophage research. A year later, working with Thomas J.
Kelly, Jr. and Kent W. Wilcox, Smith isolated and characterized the first Type II restriction
endonuclease (HindII) from Haemophilus influenzae and determined the sequence of its
cleavage site (2, 3). In recognition of this discovery, he was awarded the 1978 Nobel Prize in
Physiology or Medicine with Werner Arber and Daniel Nathans.
These studies led to Smith’s subsequent research on DNA methylases and nucleases in H.
influenzae. The two JBC Classics reprinted here detail Smith’s efforts to discover the rules
governing sequence recognition in the Type II restriction endonuclease HhaII via x-ray
crystallography. To facilitate these studies, Smith and his colleagues engineered a two-
plasmid system in Escherichia coli that overproduced HhaII on induction with isopropylthio-
galactoside (IPTG). The first paper describes the induction characteristics of the two-plasmid
overproducer clone and purification of the endonuclease. The second paper, published back-
to-back with the first, details the catalytic properties of the endonuclease. Smith used two
methods to follow the reactions: 1) gel electrophoretic analysis of nicked circular and linear
DNA products, and 2) release of 32P-labeled inorganic phosphate from specifically labeled

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HhaII sites in a reaction coupled with bacterial alkaline phosphatase. Smith’s two-plasmid
system eventually allowed him to obtain crystals of the HhaII endonuclease with a heptanucle-
otide DNA duplex (4).
Smith served on the faculty at Johns Hopkins for 30 years before retiring as American
Cancer Society Distinguished Research Professor Emeritus of Molecular Biology and Genetics
in 1998. In 1993, he accepted an appointment to the scientific advisory council of The Institute
for Genomic Research, which led to his collaboration with J. Craig Venter in the sequencing of
H. influenzae by whole genome shotgun sequencing and assembly. Five years later, Smith
joined Celera Genomics, where he was senior director of DNA Resources and aided in the
sequencing of the Drosophila and human genomes. In 2005, he co-founded Synthetic Genom-
ics, an off-shoot of Celera. He also serves as scientific director of the Synthetic Biology &
Biological Energy Groups at the J. Craig Venter Institute. In addition to the Nobel Prize,
Smith has received several honors including election to the National Academy of Sciences in
1980.1
Nicole Kresge, Robert D. Simoni, and Robert L. Hill

REFERENCES
1. JBC Classics: Brown, P. K., and Wald, G. (1956) J. Biol. Chem. 222, 865– 877 (http://www.jbc.org/cgi/
content/full/280/20/e17)
2. Kelly, T. J., Jr., and Smith, H. O. (1970) A restriction enzyme from Hemophilus influenzae. II. J. Mol. Biol. 51,
393– 409
3. Smith, H. O., and Wilcox, K. W. (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and
general properties. J. Mol. Biol. 51, 379 –391
4. Chandrasegaran, S., Smith, H. O., Amzel, M. L., and Ysern, X. (1986) Preliminary x-ray diffraction analysis of
HhaII endonuclease-DNA cocrystals. Proteins 1, 263–266
5. Smith, H. O. (1979) Hamilton O. Smith—Autobiography. Les Prix Nobel. The Nobel Prizes 1978 (Odelberg, W., ed)
Stockholm

1
Biographical information on Hamilton O. Smith was taken from Ref. 5.
The Characterization of Restriction Endonucleases: the Work of Hamilton Smith
Nicole Kresge, Robert D. Simoni and Robert L. Hill
J. Biol. Chem. 2010, 285:e2-e3.

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