Tdq

=> 5'tm

a

b e

FIGURE 10-2 Holliday junction cleavage. Two alternative pairs of DNA sites can be cut during resolution Cleavage at one pair of sites generates the "splice" or crossover products. Cleavage at the second pair of sites yields the "patch" or non-crossover products. The inset shows a Holliday |unction DNA structure Notice that the DNA is completely base-paired in this structure.

(see fate of the Ala and C/c allele markers in the Figure). These moiecules are, therefore, also known as the non-crossover products. Factors that influence the site and polarity of resolution will be discussed below.

The Double-Strand Break Repair Model More Accurately Describes Many Recombination Events

Homologous recombination is often initiated by double-stranded breaks in DNA. A common model describing this type of genetic exchange reaction is the double-stranded break-repair pathway [Figure 10-3). As with the Holliday model, this pathway starts with aligned homologous chromosomes. But in this case, the initiating event is the introduction of a double-stranded break (DSB) in one of the two DNA molecules (Figure 10-3a). The other DNA duplex remains intact. Because double-stranded DNA breaks occur relatively frequently (as we shall see below), this type of initiating event is attractive compared to the pair of aligned nicks that are proposed to initiate recombination by the Holliday model. However, the asymmetric initial breakage of the two DNA molecules in the DSB-rGpair model necessitates that later stages in the recombination process are also asymmetric, as we will see.

After introduction of the DSB, a ONA~cleaving enzyme sequentially degrades the broken DNA molecule to generate regions of single-stranded DNA (Figure IO-3b). This processing creates single-strand extensions, known as ssDNA tails, on the broken DNA molecules; these ssDNA tails terminate with 3'ends. In some cases, both strands at a DSB are processed, whereas in Other cases, only the 5'-terminating strand is degraded.

The ssDNA tails generated by this process then invade the unbroken homologous DNA duplex (Figure 10-3c). This panel of the figure shows one strand invasion, as likely occurs initially, whereas the next panel shows the two invading strands. In each case, the invading strand base-pairs with its complementary strand in the other DNA molecule. Because the invading strands end with 3' termini, they can serve as primers for new DNA synthesis. Elongation from these DNA ends-using the complementary strand in the homologous duplex as a template—serves to regenerate the regions of DNA that were destroyed during the processing of the strands at the break site (Figure 10-3 d,e).

If the two original DNA duplexes were not identical in sequence near the site of the break (for example, having single base-pair changes as described above), sequence information could be lost during recombination by the DSB-repair pathway. In the recombination event shown in Figure 10-3, sequence information lost from the gray DNA molecule as a result of DNA processing is replaced by the sequence present on the blue duplex as a result of DNA synthesis. This nonreciprocal step in DSB-repair sometimes leaves a genetic trace—giving rise to a gene conversion event—a point we will return to at the end of the chapter.

The two Holliday junctions found in the recombination intermediates generated by this model move by branch migration and ultimately arc resolved to finish recombination, Once again, the strands that are cleaved during resolution of these Holliday junctions determine whether the product DNA molecules will contain reasserted genes in the regions flanking the site of recombination (that is, result in crossing over) or not. The different ways to resolve a recombination intermediate containing two Holliday junctions are explained in Box 10-1, How to Resolve a Recombination Intermediate w ith Two Holliday Junctions.

double-stranded break (OSB>^

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