Genes VII
14.3 Double-strand breaks initiate recombination |
| Figure 14.1 Recombination occurs during the first meiotic prophase. The stages of prophase are defined by the appearance of the chromosomes, each of which consists of two replicas (sister chromatids), although the duplicated state becomes visible only at the end. The molecular interactions of any individual crossing-over event involve two of the four duplex DNAs. |
| Figure 14.2 Recombination between two paired duplex DNAs could involve reciprocal single-strand exchange, branch migration, and nicking. |
| Figure 14.5 Recombination is initiated by a double-strand break, followed by formation of single-stranded 3 F ends, one of which migrates to a homologous duplex. |
The general model of Figure 14.1 shows that a break must be made in one duplex in order to generate a point from which single strands can unwind to participate in genetic exchange. Both strands of a duplex must be broken to accomplish a genetic exchange. Figure 14.2 shows a model in which individual breaks in single strands occur successively. However, genetic exchange is actually initiated by a double-strand break. The model is illustrated in Figure 14.5.
Recombination is initiated by an endonuclease that cleaves one of the partner DNA duplexes, the "recipient." The cut is enlarged to a gap by exonuclease action. The exonuclease(s) nibble away one strand on either side of the break, generating 3′ single-stranded termini. One of the free 3′ ends then invades a homologous region in the other, "donor" duplex. The formation of heteroduplex DNA generates a D loop, in which one strand of the donor duplex is displaced. The D loop is extended by repair synthesis, using the free 3′ end as a primer.
Eventually the D loop becomes large enough to correspond to the entire length of the gap on the recipient chromatid. When the extruded single strand reaches the far side of the gap, the complementary single-stranded sequences anneal. Now there is heteroduplex DNA on either side of the gap, and the gap itself is represented by the single-stranded D loop.
The duplex integrity of the gapped region can be restored by repair synthesis using the 3′ end on the left side of the gap as a primer. Overall, the gap has been repaired by two individual rounds of single-strand DNA synthesis.
Branch migration converts this structure into a molecule with two recombinant joints. The joints must be resolved by cutting.
If both joints are resolved in the same way, the original noncrossover molecules will be released, each with a region of altered genetic information that is a footprint of the exchange event. If the two joints are resolved in opposite ways a genetic crossover is produced.
The structure of the two-jointed molecule before it is resolved illustrates a critical difference between the double-strand break model and models that invoke only single-strand exchanges.
- Following the double-strand break, heteroduplex DNA has been formed at each end of the region involved in the exchange. Between the two heteroduplex segments is the region corresponding to the gap, which now has the sequence of the donor DNA in both molecules ( Figure 14.5). So the arrangement of heteroduplex sequences is asymmetric, and part of one molecule has been converted to the sequence of the other (which is why the initiating chromatid is called the recipient).
- Following reciprocal single-strand exchange, each DNA duplex has heteroduplex material covering the region from the initial site of exchange to the migrating branch ( Figure 14.2). In variants of the single-strand exchange model in which some DNA is degraded and resynthesized, the initiating chromatid is the donor of genetic information.
The double-strand break model does not reduce the importance of the formation of heteroduplex DNA, which remains the only plausible means by which two duplex molecules can interact. However, by shifting the responsibility for initiating recombination from single Vstrand to double-strand breaks, it influences our perspective about the ability of the cell to manipulate DNA.
The involvement of double-strand breaks seems surprising at first sight. Once a break has been made right across a DNA molecule, there is no going back. Compare the events of Figure 14.2 and Figure 14.5. In the single-strand exchange model, at no point has any information been lost. But in the double-strand break model, the initial cleavage is immediately followed by loss of information. Any error in retrieving the information could be fatal. On the other hand, the very ability to retrieve lost information by resynthesizing it from another duplex provides a major safety net for the cell.