Conservative site-specific recombination is often used by DNA viruses to move their genomes in and out of the genomes of their host cells. When integrated into its host genome, the viral DNA is replicated along with the host DNA and is faithfully passed on to all descendent cells. If the host cell suffers damage (for example, by UV irradiation), the virus can reverse the site-specific recombination reaction, excise its genome, and package it into a virus particle. In this way, many viruses can replicate themselves passively as a component of the host genome, but can also “leave the sinking ship” by excising their genomes and packaging them in a protective coat until a new, healthy host cell is encountered.
Several features distinguish conservative site-specific recombination from transposition. First, conservative site-specific recombination requires specialized DNA sequences on both the donor and recipient DNA (hence the term site-specific). These sequences contain recognition sites for the particular recombinase that will catalyze the rearrangement. In contrast, transposition requires only that the transposon have a specialized sequence; for most transposons, the recipient DNA can be of any sequence. Second, the reaction mechanisms are fundamentally different. The recombinases that catalyze conservative site-specific recombination resemble topoisomerases in the sense that they form transient high-energy covalent bonds with the DNA and use this energy to complete the DNA rearrangements (see Figure 5–21). Thus, all the phosphate bonds that are broken during a recombination event are restored upon its completion (hence the term conservative). Transposition, in contrast, does not proceed through a covalently joined protein–DNA intermediate, and this process leaves gaps in the DNA that must be repaired by DNA polymerases.
Conservative Site-Specific Recombination Can Be Used to Turn Genes On or Off
Many bacteria use conservative site-specific recombination to control the expression of particular genes. A well-studied example occurs in Salmonella bacteria and is known as phase variation. The switch in gene expression results from the occasional inversion of a specific 1000-nucleotide-pair piece of DNA, brought about by a conservative site-specific recombinase encoded in the Salmonella genome. This change alters the expression of the cell-surface protein flagellin, for which the bacterium has two different genes (Figure 5–65). The DNA inversion changes the orientation of a promoter (a DNA sequence that directs transcription of a gene) that is located within the inverted DNA segment. With the promoter in one orientation, the bacteria synthesize one type of flagellin; with the promoter in the other orientation, they synthesize the other type. The recombination reaction is reversible, allowing bacterial populations to switch back and forth between the two types of flagellin. Inversions occur only rarely, and because such changes in the genome will be copied faithfully during all subsequent replication cycles, entire clones of bacteria will have one type of flagellin or the other.
Phase variation helps protect the bacterial population against the immune response of its vertebrate host. If the host makes antibodies against one type of flagellin, a few bacteria whose flagellin has been altered by gene inversion will still be able to survive and multiply.
Like many of the mechanisms used by cells and viruses, site-specific recombination has been put to work by scientists to study a wide variety of problems. To decipher the roles of specific genes and proteins in complex multicellular organisms, genetic engineering techniques are used to produce worms, flies, and mice carrying a gene encoding a site-specific recombination enzyme plus a carefully designed target DNA with the DNA sites that this enzyme recognizes. At an appropriate time, the gene encoding the enzyme can be activated to rearrange the target DNA sequence. Such a rearrangement is widely used to delete a specific gene in a particular tissue of a multicellular organism (Figure 5–66). It is particularly useful when the gene of interest plays a key role in the early development of many tissues, and a complete deletion of the gene from the germ line would cause death
Figure 5–65 Switching gene expression by DNA inversion in bacteria.