Most of us are aware of genetic engineering systems like Cre-Lox, TALENs, Zinc finger systems, and of course, CRISPR-Cas9. These are all examples of CSSR- Conservative Site-Specific Recombination. We use these site specific recombinases routinely, but do we really know about them or what the future hold for these tools?
It turns out that CSSR is yet another technique that is borrowed from the humble bacteria. Though these systems are present in both eukaryotes, prokaryotes, and phages, the ones we routinely use in genetic engineering generally come from bacteria. In particular, lambda phage uses lambda integrase to integrate into its bacterial host, and Salmonella uses Hin recombinase to express alternative genes. Furthermore, Xer recombinase catalyzes the monomerization of chromosomes in an array of bacterial species, to pre-adapt a bacterium to changes in the environment.
CSSR Features in Common
All CSSR systems have common features:
- They consist of two major elements: the enzyme, and the recombination site.
- The recombination sites have to be present in the DNA molecule(s) undergoing recombination. They provide a site for the enzyme to attack and contribute to its specificity.
- Depending on the orientation of the sites, the result of the reaction may be excision of a DNA segment, insertion of another segment, inversion within the DNA molecule, or cassette exchange between two DNA strands.
- The process is ‘conservative’ because every DNA bond broken during the process is rejoined later.
- The process involves the formation of a covalent protein-DNA intermediate.
Structurally speaking, there are two recombinase families:1
Tyrosine recombinases have a Tyr residue in their active site, whose side-chain attacks and joins to the DNA. The mechanism is known,2,3 and proceeds as follows:
- The enzyme attaches to the two DNA molecules with two subunits attaching to each of the molecules. It is important to note that the enzyme does NOT have a four-fold symmetry—two diagonally opposite subunits are in the active conformation at any time.
- Tyr residues of the active subunits attach to the 3’-phosphate group of one strand in their own DNA molecule (first strand cleavage), leaving a free 5’-OH group.
- The 5’-OH groups attach to the exposed phosphate group of the OTHER molecule, forming a Holliday junction. Now, one strand has recombined with the homologous strand in the other molecule, while the other strand in both molecules remains untouched.
- Now, the opposite subunits of the enzyme switch to the active conformation while the old active ones become inactive. These new subunits attack the untouched strand of the DNA molecule (second strand cleavage), leaving an exposed 5’-OH end while attaching to the 3’-phosphate group.
- The process of attaching of 5’OH to 3’-phosphate of the different molecule repeats, and there you have it! The two molecules have undergone recombination.
Serine recombinase systems possess a Ser residue in the active site which binds to the DNA. The chief difference in the mechanism of action is that unlike Tyr recombinases, Ser recombinases cleave both the strands of the molecules together, and then join them to the opposite molecule’s strands.
- Two subunits pair to each DNA molecule- one subunit for each strand. All the subunits are simultaneously active as the Ser residues attack the 5’-phosphate group on each strand.
- All four strands are broken, resulting in four 3’-OH groups which can attack the corresponding strands of the different molecules.
- The protein structure shifts, resulting in swapping partners and alignment of 3’-OH and 5’-phosphate groups of different molecules with each other.
- The DNA strands then join simultaneously to yield recombinant molecules.
Applications and Future Potential
Of the recombinases in use, the majority are Tyr recombinase systems, such as Cre-Lox recombination systems. These help you to target gene knockouts, or to ‘seed’ embryonic cells with specific recombination sites, that serve as a target for the enzyme later in life. This helps scientists study genes whose knockouts may prove lethal during the embryonic stages of normal development. See here for more uses of Cre-Lox recombination. Other than Cre-Lox, FLP recombinases, are also widely in use for biotechnological applications.
In comparison, serine recombinases, such as φ C31-Int are useful in transgenic applications. This is because of their propensity for a recognizing a single recombination site which the enzyme locks onto geometrically. However, what’s rapidly becoming an exciting field is the use of Ser recombinases in synthetic biology, specifically in engineering expression logic circuits in E. coli4. Recent studies have shown how all the standard Boolean logic operations can be by the combined action of just two orthogonal serine integrases (φ C31 Int and Bxb1 Int). Furthermore, chimeric serine recombinases, in which the targeting uses improved TALE architecture, have already been reported.
In conclusion, one might wonder if ubiquitous site-specific recombination systems will become the main engines of a cell circuitry driven era of biotechnology?
- Watson JD, Baker TA, Bell SP, Gann A, Levine M, Losick R. Molecular Biology of the Gene. 7th ed. San Francisco: Pearson education; 2014.
- Grindley ND, Whiteson KL, Rice PA. (2006) Mechanisms of site-specific recombination. Annu Rev Biochem. 75:567–605.
- Jayaram M, Ma C, Kachroo AH, Rowley PA, Guga P, Fan H, Voziyanov Y. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective. In Craig NL, Chandler M, Gellert M, Lambowitz AM, Rice PA, Sandmeyer SB, editors. Mobile DNA III.Washington, DC: ASM Press; 2015. pp. 43–71.
- Stark W. The Serine Recombinases. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S, editors. Mobile DNA III. Washington, DC.:ASM Press; 2015. pp. 73–89.