The Characterization of Restriction Endonucleases: the Work of Hamilton Smith

Abstract

Purification of the HhaII Restriction Endonuclease from an Overproducer Escherichia 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

(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, because 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 science teacher who allowed him to complete chemistry and physics during the summer.

Hamilton O. Smith

After finishing high school, Smith enrolled at the University of Illinois, majoring 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 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 isopropylthiogalactoside (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 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 heptanucleotide 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 Genomics, 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