Restriction endonucleases and ligases are essential tools in cloning
To insert a given DNA sequence into a vector, one must first cleave the vector, allow recombination, and then ligate the recombinant vector. Two classes of DNA enzymes are essential for creating any recombinant DNA molecule: restrictases and ligases. DNA polymerases constitute another very important class of DNA enzymes used to manipulate nucleic acids, but as they encompass a wide variety of enzymes—each with its own role and mechanism of action—we describe them in relevant chapters elsewhere in the book.
The discovery of restriction endonucleases, also called restrictases, is one of the most important developments in molecular biology; for a historic account, see Box 5.1. The majority of these enzymes catalyze the cleavage of DNA at specific nucleotide sequences, producing defined double-stranded DNA fragments suitable for numerous applications in basic research and biotechnology.
More than 3,000 restrictases have been described to date. They are all DNA endonucleases, that is, they hydrolyze internal phosphodiester bonds within the polynucleotide chain (Figure 1). Exonucleases, by contrast, hydrolyze the chain from either its 3′- or 5′-end, removing one nucleotide at a time. The name of each restrictase carries information about the bacterial species and strain in which it was originally discovered; a Roman numeral is used to differentiate between several enzymes from the same strain. For example, EcoRI designates one of the two known restrictases in Escherichia coli strain R; HaeIII (see Figure 1) denotes the third enzyme present in Haemophilus aegyptius. There are three general classes of restrictases. Type II enzymes are of primary interest to us because of their ability to both recognize a particular DNA site and to cut at a defined point within that site, making them ideal tools for recombinant DNA technology.
Fig1. Cleavage action of restriction endonucleases. (A) In general, endonucleases that cleave polynucleotide chains internally can be specific with respect to which ester bond in the phosphodiester linkage they cleave, thus producing DNA fragments of different termini, as depicted. Type II restriction enzymes produce 5′-phosphates and 3′-OH groups only. (B) Cleavage sites on the two DNA strands can be offset, by as many as four nucleotides, or occasionally even more, to produce overhangs, also known as sticky ends. When there is no offset of the cutting positions, blunt-end products are created.
Each restriction enzyme is characterized mainly by the specific sequence it recognizes and the site where it cuts the double helix (Table 1). Most recognition sequences are 4, 5, or 6 bp in length, although longer sites have also been described. The longer the recognition site, the less frequently it occurs in DNA. For instance, enzymes that target 4-bp sites cut about once every 256 or 44 nucleotide pairs, whereas 6-bp sites will be found on average once in 4,096 or 46 nucleotide pairs. As illustrated in Figure 1A, the enzyme can attack either one of the two ester bonds in the phosphodiester linkage, producing different products. In addition, different enzymes can cut the opposite strands of the DNA helix, either in a staggered way to produce over hangs, also known as sticky ends, or exactly opposite each other to produce blunt ends, also known as flush ends (see Figure 1B). Both kinds of products can be ligated to form uninterrupted molecules, but the ligation efficiency of blunt-ended fragments is much lower than that of fragments containing overhangs, because the latter can actually base-pair with each other, increasing the probability of the two ends staying in close proximity and in proper orientation for ligation to occur.
Table1. Classification and properties for restriction/modification systems. All enzymes require Mg2+ for cleavage; only type I enzymes require ATP for functioning of the motor. The letter N stands for any nucleotide.
Type II restriction endonucleases have proven to be of great value in recombinant technologies, as they possess well-defined specificity for the cleavage sequence, and thus the products of their action are very well defined. These enzymes usually recognize palindromic sequences, which read the same on both complementary DNA strands, 5′ → 3′ and 3′ → 5′, and bind as homodimers. The cleavage patterns of type II restrictases can be rather diverse. Figure 2 illustrates some frequent cleavage patterns and introduces important concepts: isoschizomers, methylation-sensitive isoschizomers, and isocaudomers.
Fig2. Diversity of restriction endonuclease cleavage patterns. (A) Ambiguity of recognition sequences, as illustrated for the example of HindII, the first type II enzyme to be described. Py stands for either pyrimidine, C or T; Pu stands for either purine, A or G. All possible sequences of this kind are cleaved by HindII. (B) Isoschizomers are enzymes from different sources that recognize the same target. Some pairs of isoschizomers cut their targets at different places, as in the example shown. (C) Methylation-sensitive isoschizomers: if the recognition sequence is methylated, in this case on the C, one enzyme of the isoschizomer pair will cut it while the other one will not. In the specific example shown, HpaII is methylation-sensitive and will not cut when C is methylated, while MspI will cut whether C is methyl ated or not; that is, MspI is methylation-indifferent. Such pairs of enzymes are extensively used in research to probe the methylation status of a particular sequence. (D) Isocaudomers: production of a hybrid site by cohesion of complementary sticky ends generated by two differ ent enzymes. One of these enzymes (Sau3AI) recognizes a 4-bp sequence that is embedded in the hexanucleotide sequence recognized by a different enzyme, BamHI in this case. The sticky ends produced by these enzymes can base-pair to produce a hybrid site. The new site is sensitive to Sau3AI but may not constitute a target for BamHI, depending on the nucleotides adjacent to the original Sau3AI site.
Isoschizomers are pairs of restriction enzymes that have the same recognition site but come from different bacterial species; thus, they may require different reaction conditions. Isoschizomers may cut the sequence at different places, thus adding to the diversity of cleavage patterns and DNA ends produced (see Figure 2B for an example). The first enzyme discovered to recognize a given sequence is known as the prototype; all subsequently identified enzymes that recognize that same sequence are isoschizomers. Methylation-sensitive isoschizomers (see Figure 2C) may or may not cleave a sequence, depending on whether the sequence carries methyl groups. These pairs of enzymes have proven instrumental in studying the methyla tion patterns of specific sequences of interest. Finally, isocaudomers are pairs of restriction enzymes that have slightly different recognition sequences, but generate identical overhanging termini sequences upon cleavage of DNA (see Figure 2D). These sequences can be ligated to one another and form an asymmetrical sequence that can or cannot be cleaved by a restriction enzyme, depending on the adjacent sequence.
The initial binding of type II restrictases occurs at random sites, and the subsequent search for the recognition sequence involves one-dimensional translocation along the DNA. Numerous restrictases have been crystallized and their structures have been determined in their free forms, bound to noncognate (nonspecific) DNA fragments, and bound to cognate sequences. Figure 3 compares the crystal structures of EcoRV when bound to a random sequence and when bound to its specific recognition site. Note that interaction with the cognate nucleotide sequence leads to conformational changes in the enzyme that allow it to embrace the DNA more tightly.
Fig3. Comparison of crystal structures of nonspecific and specific enzyme DNA complexes for EcoRV. (A) The nonspecific enzyme–DNA complex presumably closely reflects the structure of the complex that slides along DNA during the initial one-dimensional search for the recognition sequence. (B) The specific enzyme–DNA complex crystallized in the presence of Ca2+ can be considered to resemble the enzyme-substrate complex. Note the conformational transitions in some of the helices that embrace the DNA once the enzyme recognizes the sequence to be cleaved. [From Pingoud A, Fuxreiter M, Pingoud V et al. (2005) Cell Mol Life Sci 62: 685–707. With permission from Springer Science and Business Media.]
Type I restrictases are multifunctional enzymes that combine both nuclease and methylase activity in a single protein trimeric complex (Figure 4). One subunit, R, carries the restriction function, cleaving the DNA at random locations, sometimes very far, 10 kilobase pairs or more, from the enzyme’s recognition sequence; it also has an ATP-dependent motor activity that is involved in translocating the DNA. The second subunit, M, carries out the DNA methylation reaction, which occurs in the recognition site itself. The third subunit, S, recognizes the specific sequence to which the complex binds. For cleavage to occur, the recognition/binding site has to be brought into proximity with the cleavage site; that is, the intervening DNA has to loop out (at the same time it is supercoiled) (see Figure 4). Therefore, these enzymes are unusual molecular motors that bind specifically to DNA and then move the rest of the DNA through this bound complex.
Fig4. Structure and activity of EcoR124I, a type I restrictase. (A) The enzyme, with subunit composition R2 M2 S, possesses three functionally dis tinct subunits. The S subunit is responsi ble for DNA binding specificity, while M is required for DNA methylation, that is, modification activity. R, along with the core M, is absolutely required for DNA cleavage, that is, restriction activity. It is also responsible for ATP binding, hydro lysis, and DNA translocation; that is, it is a molecular motor. The R subunit also possesses helicase activity and domains responsible for assembly of the com plex. (B) Successive steps in the motor activity of EcoR124I. The orange block represents the DNA binding/recognition site. When the motor complex binds to the DNA at the recognition site, it also attaches to adjacent DNA sequences. Then the motor begins to translocate these adjacent sequences through itself, following the helical path of the DNA in the complex. This leads to the creation of an expanding loop of positively super coiled DNA. During translocation, the entire complex remains tightly bound to the recognition sequence. The process is bidirectional, with each R subunit acting as a molecular motor, and proceeds until blocked by some external event, usually another enzyme also translocating the DNA; other blockages include DNA topology. Cleavage follows blockage of the translocation; the site of cleav age is random because translocation blocking is a random process. [Adapted from Pennadam SS, Firman K, Alexander C et al. (2004) J Nanobiotechnol 2: 10.1186/1477-3155-2-8. With permis sion from BioMed Central.]
Type III restriction endonucleases are very similar to type I, but they do not require ATP, they methylate just one of the strands, and their cleavage site is relatively close to the recognition sequence.
DNA ligases are also among the most important enzymes in the cell. They are indispensable for numerous cellular processes, including DNA replication, recombination, and repair whenever there is a need to seal nicks in the DNA double helix or join broken helices. Of interest to us here is their essential role in joining fragments of double-helical DNA to form intact molecules in recombinant DNA technologies. Ligases form phosphodiester bonds between the 3′-OH and 5′-phosphate termini of DNA fragments (Figure 5A). The reaction involves several steps. Adenylation of the active-site lysine uses ATP in phages and eukaryotes but uses NAD+, nicotinamide adenine dinucleotide, in bacteria. Figure 5B and Figure 5C depict the domain structure of human ligase I and the crystal structure of this enzyme complexed with DNA.
Fig5. DNA ligation. (A) Steps in the reaction catalyzed by DNA ligase. (Step 1) Enzyme-AMP is formed by attack of lysine on the α-phosphate of ATP, releasing inorganic pyrophosphate. (Step 2) The 5′-phosphate of the nicked DNA strand, downstream side, attacks the Lys-AMP intermediate to form an App-DNA intermediate, where pp indicates a pyrophosphate linkage, 5′-P to the 5′-phosphate of AMP. (Step 3) The 3′-OH end of the nicked strand, upstream side, attacks the 5′-P of App-DNA, covalently joining the DNA strands and liberating AMP. Thus, ligation pays an energy price of one ATP. (B) Domain organization of human ligase I. OB-fold is the oligonucleotide/oligosaccharide-binding domain. (C) Left, the three domains, colored as in part B, fully encompass the App-DNA reaction intermediate. The intact DNA strand is shown in black, the nicked strand is shown in gray, and the App-DNA linkage is shown in blue. Gray spheres indicate a poorly ordered surface loop at residues 385–392. Right, molecular surface: the adenylation domain, AdD, is semitransparent to highlight the AMP cofactor held within its active site. [Adapted from Pascal JM, O’Brien PJ, Tomkinson AE et al. (2004) Nature 432: 473–478. With permission from Macmillan Publishers, Ltd.]
For the purposes of recombinant DNA technology, a distinction should be made between ligases that can only join DNA ends on overhangs and those that can ligate blunt-end DNA fragments. Most use is made of the first type: it is mechanistically easier to join the phosphodiester backbones of fragments that are kept in close proximity by base pairing of complementary overhangs. The E. coli DNA ligase is a representative of this type. Joining together blunt ends is much more challenging, and the enzymes that are capable of doing this are much less efficient. An example of such an enzyme is the DNA ligase from bacteriophage T4. The efficiency of blunt-end ligation can be increased by using small oligonucleotides that carry restriction sites for sticky-end production, known as DNA linkers. The strategy for using linkers is depicted in Figure 6.
Fig6. DNA linkers for joining of blunt-end DNA fragments. Linkers can be chemically synthesized to contain any restriction target site. Blunt-end ligation is performed by the DNA ligase from phage T4, as the E. coli DNA ligase will not catalyze blunt-end ligation except under special reaction conditions of macromolecular crowding. Linkers can be ligated to both ends of the blunt-end DNA fragment to be cloned; upon treatment with the restriction endonuclease that recognizes the linker sequence, sticky ends are produced. This treatment is then followed by standard cloning techniques that use sticky ends for efficient ligation of the foreign gene into a vector treated with the same enzyme.
The availability of site-specific endonucleases and DNA ligases made it possible to create the first recombinant DNA molecules in the early 1970s (Box 5.2).
