Molecular Biotechnology. Bernard R. Glick

Generates Cohesive Ends

      Recombinant DNA technology requires a vector to carry cloned DNA, the specific joining of vector and cloned (insert) DNA molecules to form a vector–insert DNA construct, the introduction of the vector–insert DNA construct into a host cell, and the identification of host cells that acquire the cloned DNA. The discovery of type II restriction endonucleases facilitated cloning genes into vectors. In 1968, M. Meselson and R. Yuan (Nature 217:1110–1114) showed that the capability of a strain of E. coli to prevent (restrict) the development of a bacterial virus (bacteriophage) was due to a host cell enzyme that cleaved the DNA of the infecting bacteriophage. The study done by Mertz and Davis (Proc. Natl. Acad. Sci. USA. 69:3370–3374, 1972) established that the RI restriction endonuclease from E. coli, which is now called EcoRI, cut DNA at a specific site and produced complementary extensions. Briefly, they showed that after circular DNA was linearized by treatment with EcoRI, some of the molecules formed hydrogen-bonded circular DNA molecules which were converted to covalently closed circular DNA molecules by treating the sample with a DNA ligase. The extensions of all of the cut DNA molecules were the same and were estimated to be four to six nucleotides long, with the recognition site being six nucleotide pairs. Mertz and Davis concluded that “… any two DNA molecules with RI sites can be ‘recombined’ at their restriction sites by the sequential action of RI endonuclease and DNA ligase to generate hybrid DNA molecules.” The discovery that EcoRI created cohesive ends was one of the most important contributions to the development of recombinant DNA technology because it provided, according to Mertz and Davis, a “simple way … to generate specifically oriented recombinant DNA molecules in vitro.”

      A large number of restriction endonucleases from different bacteria are available to facilitate cloning. The sequence and length of the recognition site in the DNA vary among the different enzymes and can be four or more nucleotide pairs (Table 2.1). One of the first restriction endonucleases to be characterized was from the bacterium Escherichia coli, designated EcoRI. The name of a restriction endonuclease indicates the genus (capitalized letter), species (first two letters in lowercase), and occasionally the strain or serotype (e.g., R in EcoRI) of the source bacterium, as well as the order of characterization of different restriction endonucleases from the same bacterium (Roman numerals). As for most restriction endonucleases, EcoRI is a homodimeric protein (made up of two identical polypeptides) that recognizes and binds to a specific, palindromic DNA sequence (Fig. 2.1A). In DNA, a palindrome is a sequence of nucleotides in each of the two strands that is identical when either is read in the same polarity, i.e., 5′ to 3′. The EcoRI recognition sequence consists of six base pairs (bp) and is cut between the guanine and adenine residues on each strand (Fig. 2.1A). Specifically, it cleaves the bond between the oxygen attached to the 3′ carbon of the sugar of one nucleotide and the phosphate group attached to the 5′ carbon of the sugar of the adjacent nucleotide. The symmetrical staggered cleavage of DNA by EcoRI produces two single-stranded, complementary ends, each with extensions of four nucleotides, often referred to as sticky ends. Each single-stranded extension terminates with a 5′ phosphate group, and the 3′ hydroxyl group of the opposite strand is recessed (Fig. 2.1A). Some other restriction endonucleases, such as PstI, leave 3′ hydroxyl extensions with recessed 5′ phosphate ends (Fig. 2.1B), while others, such as SmaI, cut the backbone of both strands within a recognition site to produce blunt-ended DNA molecules (Fig. 2.1C).

Table 2.1 Recognition sequences of some restriction endonucleases

      Figure 2.1 Type II restriction endonucleases bind to and cut within a specific DNA sequence. (A) EcoRI makes a staggered cut in the DNA strands producing single-stranded, complementary ends (sticky ends) with a 5′ phosphate group extension; (B) PstI also makes a staggered cut in both strands but produces sticky ends with a 3′ hydroxyl group extension; (C) cleavage of DNA with SmaI produces blunt ends. Arrows show the sites of cleavage in the DNA backbone. S, deoxyribose sugar; P, phosphate group; OH, hydroxyl group. The restriction endonuclease recognition site is shaded.

      Restriction endonucleases isolated from different bacteria may recognize and cut DNA at the same site (Fig. 2.2A). These enzymes are known as isoschizomers. Some recognize and bind to the same sequence of DNA but cleave at different positions (neoschizomers), producing different single-stranded extensions (Fig. 2.2B). Other restriction endonucleases (isocaudomers) produce the same nucleotide extensions but have different recognition sites (Fig. 2.2C). In some cases, a restriction endonuclease will cleave a sequence only if one of the nucleotides in the recognition site is methylated, while in other cases a restriction endonuclease (type IIS) binds to a specific recognition site but cuts outside of that site, a fixed number of nucleotides away from one or both ends. In the latter case, any particular sequence of nucleotides may be present between the binding sequence and the cut sites. These characteristics of restriction endonucleases are considered when designing a cloning experiment.

      Figure 2.2 Restriction endonucleases have been isolated from many different bacteria. (A) Isoschizomers such as BspEI from Bacillus sp. and AccIII from Acinetobacter calcoaceticus bind the same DNA sequence and cut at the same sites; (B) neoschizomers such as NarI from Nocardia argentinensis and SfoI from Serratia fonticola bind the same DNA sequence but cut at different sites; (C) isocaudomers such as NcoI from Nocardia corallina and PagI from Pseudomonas alcaligenes bind different DNA sequences but produce the same sticky ends. Bases in the restriction enzyme recognition sequence are shown. Arrows show the sites of cleavage in the DNA backbone.

      Many other enzymes may be used to prepare DNA for cloning. In addition to restriction endonucleases, nucleases that degrade single-stranded extensions, such as S1 nuclease and mung bean nuclease, are used to generate blunt ends for cloning (Fig. 2.3A). This is useful when the recognition sequences for restriction enzymes that produce complementary sticky ends are not available on both the vector and target DNA molecules. Blunt ends can also be produced by extending 3′ recessed ends using a DNA polymerase such as Klenow polymerase derived from E. coli DNA polymerase I (Fig. 2.3B). Phosphatases such as calf intestinal alkaline phosphatase cleave the 5′ phosphate groups from restriction enzyme-digested DNA (Fig. 2.3C). A 5′ phosphate group is required for formation of a phosphodiester bond between nucleotides, and therefore, its removal prevents recircularization (self-ligation) of vector DNA. On the other hand, kinases add phosphate groups to the ends of DNA molecules. Among other activities, T4 polynucleotide kinase catalyzes the transfer of the terminal (γ) phosphate from a nucleoside triphosphate to the 5′ hydroxyl group of a polynucleotide (Fig. 2.3D). This enzyme is employed to prepare chemically synthesized DNA for cloning, as such DNA molecules are often missing a 5′ phosphate group required for ligation to vector DNA.

      Figure 2.3 Some other enzymes used to prepare DNA for cloning. (A) Mung bean nuclease degrades single-stranded 5′ and 3′ extensions to generate blunt ends; (B)