35.3 DNA Recombination Plays Important Roles in Replication and Repair

Figure 35.14: Generation of a double-strand break. DNA replication takes place at the replication fork. When the replication machinery on one strand encounters a nick in the DNA, replication stalls. The replication fork may collapse, yielding an uncompleted strand with a double-strand break.

Most processes associated with DNA replication function to copy the genetic message as faithfully as possible. However, several biochemical processes require the recombination of genetic material between two DNA molecules. In genetic recombination, two daughter molecules of DNA are formed by the exchange of genetic material between two parent molecules. Under what circumstance will recombination be required for DNA repair?

Common DNA lesions that can be repaired by recombination are breaks in both strands of a DNA helix. Double-strand breaks arise when replication stalls, such as when the polymerase encounters an unrepaired nick in one of the template strands at the replication fork (Figure 35.14). The replication fork collapses, leaving a double-strand break on one of the daughter helices. Double-strand breaks can also result from exposure to ionizing radiation. Radiation on the short-wavelength end of the electromagnetic spectrum—notably, x-ray and gamma rays—is powerful enough to break the DNA backbone. Let’s examine how a double-strand break caused by ionizing radiation is repaired by recombination.

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Recombination is most efficient between stretches of DNA that are similar in sequence. In homologous recombination, parent DNA duplexes align at regions of sequence similarity, and new DNA molecules are formed by the breakage and joining of homologous segments.

Double-Strand Breaks Can Be Repaired by Recombination

Recombination is a complex process, requiring dozens of proteins. One key protein in recombination in humans is RAD51, an ATPase that binds single-stranded DNA. How are single-stranded regions generated at the site of a double-strand break?

The repair process begins with the digestion of the 5′ ends at the break site, generating single-stranded regions of DNA that are then bound by multiple copies of RAD51. The single-stranded DNA then displaces one of the strands of the undamaged double helix in an ATP-dependent process called strand invasion. The resulting three-stranded structure is called a displacement loop or D-loop. DNA synthesis takes place, with the use of the undamaged helix as a template. A second strand invasion then takes place to complete the repair of the damaged strand, resulting in the formation of two crosslike structures called Holliday junctions (Figure 35.15). Cleavage of the Holliday junctions, followed by ligation, yields two intact DNA double helices.

Figure 35.15: Repair of double-strand break by using recombination. 1. A 5′ exonuclease generates single-strand DNA at the site of the break. 2. Strand invasion takes place when the strand on the damaged DNA base-pairs with the complementary strand on the undamaged DNA, forming a displacement loop, or D-loop. 3. DNA synthesis takes place. 4. A second strand invasion takes place, resulting in the formation of two Holliday junctions. 5. The Holliday junctions are cleaved and ligated, forming two intact molecules of DNA.

DNA Recombination Is Important in a Variety of Biological Processes

In the example illustrating the use of recombination for DNA repair, we assumed for simplicity’s sake that the DNA sequences on the homologous strands were identical. However, such identical sequences are rarely the case. There are sequence differences—ranging from slight to substantial—in the alleles of the same gene on homologous DNA molecules. Consequently, when recombination takes place, new DNA sequences are generated. In meiosis, the limited exchange of genetic material between paired chromosomes provides a simple mechanism for generating genetic diversity in a population. Recombination also plays a crucial role in generating molecular diversity for antibodies and some other molecules in the immune system. Some viruses employ recombination pathways to integrate their genetic material into the DNA of a host cell.

DID YOU KNOW?

Meiosis is a special type of cell division that is required for sexual reproduction. In animals, meiosis results in the production of sperm and eggs.

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Recombination is the foundation for an enormously powerful biochemical tool. Recombination is used to move, excise, or insert genes in, for example, the generation of “gene-knockout” mice, in which a specific gene is deleted, or “knock-in” mice, in which a specific gene is inserted. Such modified mice have proved to be valuable experimental tools.

For example, the gene-knockout approach has been applied to the genes encoding transcription factors that control the differentiation of muscle cells. When both copies of the gene for the regulatory protein myogenin are disrupted, an animal dies at birth because it lacks functional skeletal muscle. Microscopic inspection reveals that the tissues from which muscle normally forms contain precursor cells that have failed to differentiate fully (Figure 35.16). Heterozygous mice containing one normal gene for myogenin and one disrupted gene appear normal, suggesting that the level of gene expression is not essential for its function. Analogous studies have probed the function of many other genes to generate animal models for known human genetic diseases.

!quickquiz! QUICK QUIZ 2

How many strands of DNA are present in a Holliday structure?

Figure 35.16: Consequences of gene disruption by recombination. Sections of muscle from normal (A) and gene-disrupted-by-recombination (B) mice, as viewed under the light microscope. Muscles do not develop properly in mice having both genes for myogenin disrupted. Arrows indicates bones of the pelvis, establishing that the two views show the same anatomical region. M in part B indicates a muscle fiber in the knockout mouse.