In its simplest form, this type of base-pairing interaction can be mimicked in a test tube by allowing a DNA double helix to re-form from its separated single strands. This process, called DNA renaturation or hybridization, occurs when a rare random collision juxtaposes complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix-nucleation step is followed by a very rapid “zippering” step, as the region of double helix is extended to maximize the number of base-pairing interactions (Figure 5–47).
DNA hybridization can create a region of DNA double helix consisting of strands that originate from two different duplex DNA molecules as long as they are complementary, or nearly so. As we will see shortly, the formation of such a hybrid molecule, known as a heteroduplex, is an essential feature of homologous recombination. DNA hybridization and heteroduplex formation is also the basis for many of the methods used to study cells, and we will discuss these uses in Chapter 8.
The DNA in a living cell is almost all in the stable double-helical form, so the reaction depicted in Figure 5–47 rarely occurs in vivo. Instead, as we shall see, homologous recombination is brought about through a carefully controlled set of reactions that allow two DNA duplexes to sample each other’s sequences without fully dissociating into single strands.
We saw in the previous section that nonhomologous end-joining occurs without a template and usually leaves a mutation at the site at which a double-strand break is repaired. In contrast, homologous recombination can repair double-strand breaks accurately, without any loss or alteration of nucleotides at the site of repair. For homologous recombination to do this repair job, the broken DNA has to be brought into proximity with homologous but unbroken DNA, which can serve as a template for repair. For this reason, homologous recombination often occurs just after DNA replication, when the two daughter DNA molecules lie close together and one can serve as a template for repair of the other. As we shall see, the process of DNA replication itself creates a special risk of accidents requiring this sort of repair.
The simplest pathway through which homologous recombination can repair double-strand breaks is shown in Figure 5–48. In essence, the broken DNA duplex and the template duplex carry out a “strand dance” so that one of the damaged strands can use the complementary strand of the intact DNA duplex as a template for repair. First, the ends of the broken DNA are chewed back, or “resected,” by specialized nucleases to produce overhanging, single-strand 3ʹ ends. The next step is strand exchange (also called strand invasion), during which one of the single-strand 3ʹ ends from the damaged DNA molecule worms its way into the template duplex and searches it for homologous sequences through base-pairing. We describe this remarkable reaction in detail in the next section. Once stable base-pairing is established (which completes the strand exchange step), an accurate DNA polymerase extends the invading strand by using the information provided by the undamaged template molecule, thus restoring the damaged DNA. The last steps—strand displacement, further repair synthesis, and ligation—restore the two original DNA double helices and complete the repair process. Homologous recombination resembles other DNA repair reactions in that a
Figure 5–47 DNA hybridization. DNA double helices can re-form from their separated strands in a reaction that depends on the random collision of two complementary DNA strands. The vast majority of such collisions are not productive, as shown on the left, but a few result in a short region where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to the formation of a complete double helix. Through this trial-and-error process, a DNA strand will find its complementary partner even in the midst of millions of nonmatching DNA strands.
DNA polymerase utilizes a pristine template to restore damaged DNA. However, instead of using the partner complementary strand as a template, as occurs in most DNA repair pathways, homologous recombination exploits a complementary strand from a separate DNA duplex.