Box 10-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions

How the Holliday junctions present rn a recombination inter mediate ere cleaved has a huge impact on the structure of the product DNA molecules. Products will either have the DMA flanking the site of recombination reassorted (in the splice/crossover products) or not (in the patch/non-crossover products) depending on how resolution is achieved. Because the intermediates generated by the DSB-repair pathway contain two Holliday ¡unctions, it can be difficult to see which products are generated by the different possible combinations of Holliday junction cleavage events. In fact, there is a simple pattern that determines whether crossover or non-crossover products are generated.

To explajn the different possible ways these intermediates can be resolved, consider the two junctions (labeled x and y) in Box 10-1 Figure 1 For each junction, there are two possible cleavage sites (labeled site 1 and site 2). The simple rule that determines whether or not resolution will result in crossover versus non-crossover products is as follows. If both junctions are cleaved in the some way, that is either both at site 1 or both at site 2, then non-crossover products will be generated. An example of this type of product is shown in panel b of the figure; these are the molecules generated when both Holliday junctions are cleaved at site 2. Notice, the allele markers A/B and a/b are still on the same DNA molecules as they were in the parental chromosomes. Cleavage of both junctions at site I also generates non-crossover products.

In contrast, when the two Holliday junctions are cleaved using different sjtes, then the crossover products are generated. An example of this type of resolution is shown in panel c of Box 10-1 Figure 1. Here junction x was deaved at site t whereas junction y was cleaved at site 2. Notice that now gene A is linked to gene b, whereas gene a is linked to gene B, thus reassortment of the flanking genes has occurred. Cleavage of ¡unction x at site 2 and junction y at site 1 also generates crossover products.

Why is the simple rule true? To understand this, compare the junctions shown here to the single Holliday junction shown in Figure 10-2 You should see that, at a single junction, cleavage at site t would give the splice products, whereas cleavage at site 2 would generate patch products So when you combine the results of cleavage at the two junctions, this is what happens:

• Cleavage of both junctions at site 7 will give a patch product (patch + patch = patch, non-crossover products).

• Cleavage at both junctions at site 1 also gives a patch product (splice + splice = patch because the second splice-type resolution essentially "undoes" the rearrangement caused by the first cleavage).

• Cleavage of one junction at site I, but the other at site 7 therefore generates crossover products (splice +■ patch = splice), because the rearrangement caused by the site ) cleavage is retained in the final product juncttonx ®

juncttonx ®

non-crossover products crossover products

BOX 10*1 FIGURE 1 Two possible ways of resolving an intermediate from the DSB-repair pathway. The parental OKA molecules were like those tn Figure 10-3 The regions of red DNA are those that were resynthesized during recombination.

non-crossover products crossover products

BOX 10*1 FIGURE 1 Two possible ways of resolving an intermediate from the DSB-repair pathway. The parental OKA molecules were like those tn Figure 10-3 The regions of red DNA are those that were resynthesized during recombination.

Double-Stranded DNA Breaks Arise by Numerous Means and Initiate Homologous Recombination

Dotible-slranded breaks in DNA arise quite frequently. If these breaks are not repaired, the consequence to the cell is disastrous. For example, a single DSB in the E. coli chromosome is lethal to a cell that lacks the ability to repair it. The major mechanism used to repair DSBs in most cells is homologous recombination via the DSB-repair pathway described above. Some cells also use a simpler mechanism, called nonhomologous end joining (NHEJ) as well. This process is described in Chapter 9.

In bacteria, the major biological role of homologous recombination is to repair DSBs. These broken DNA ends arise from several causes (see Chapter 9). Ionizing radiation and other damaging agents sometimes directly break both strands of the DNA backbone. Many types of DNA damage also indirectly give rise to DSBs by interfering with the progress of a replication fork. For example, an unrepaired nick in one DNA strand will lead to collapse of a passing replication fork (Figure 1D-4).

nick in template strand fork collapse

fork collapse chromosomal replication fork chromosomal replication fork lesion lesion

DNA lesion in template strand

.DSB for recombination fork collapse

FIGURE 10-4 Damage in the DNA template can lead to DSB formation during DNA replication. This is easiest to see when the template contains a ntck (left panel), but also can occur when the tern plate carnes a fork stopping lesion (right panel). In this case, the two newly syritttested strands (shown in red) can base-pair and the fork can regress. This structure can be further processed by a number of means. The broken end can serve to initiate recombination

fork regression

DSB for recombination

DSB for recombination

fork regression

Similarly, a lesion in DNA that makes a strand unable to serve as a template will stop a replication fork. This type of stalled fork can be processed by several different means (for example, fork régression or nuclease digestion; see Figure 10-4) that give rise to a DNA end with a DSH. These broken DNA ends then initiate recombination with a homologous DNA molecule, a process which will, in turn, heal the break.

In addition to repairing DSBs in chromosomal DNA, homologous recombination promotes genetic exchange in bacteria. This exchange occurs between the chromosome of one cell and DNA that enters that cell via phage-mediated transduction or cell-cell conjugation (see Chapter 21). In these cases, the entering DNA comes into the cell as a linear molecule, and thus provides the critical "broken" DNA end needed to initiate recombination.

In eukaryotic cells, homologous recombination is critical for repairing DNA breaks and collapsed replication forks, flow ever, there are other times when recombination is also needed. As we will describe below, recombination is essential to the process of chromosome pairing during meiosis. in this case, as ceils enter meiosis they produce a specific protein to introduce DSBs into the DNA and therefore initiate this recombination pathway (see below). Thus, although they arise from many different sources, the appearance of a DSB in DNA is a key early event in homologous recombination.

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