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Modern Genetic Analysis Chapter 5. Recombination of Genes
The Mechanism of Crossing-Over

Crossing-over is a remarkably precise process. Some kind of cellular machinery takes two huge molecular assemblages (homologous nonsister chromatids), breaks them in the same relative position, and then rejoins them in a new arrangement so that no genetic material is lost or gained in either. The exact molecular mechanism of crossing-over is not known, but several models have been proposed, and there is general agreement that a key step is the formation of heteroduplex DNA, a hybrid type of DNA molecule that is composed of a strand from one parental chromatid and a strand from the other.

Most of the evidence in favor of a heteroduplex model comes from the study of fungal tetrads and octads. Octads are particularly informative in pointing to the existence of heteroduplexes in crossing-over. We have seen in Chapter 4 that in these organisms a cross A × a will create a monohybrid meiocyte A/a that is expected to segregate in a 1:1 ratio in the meiotic products according to the law of equal segregation. Indeed this is found in most meiocytes. In fungi with octads the four nuclei that represent the four products of meiosis undergo an extra mitotic division to produce four ascospore pairs, which stay together in the octad sac. The expected octad ratio from a monohybrid meiocyte is 4:4. However, in rare meiocytes (generally on the order of 0.1 percent to 1 percent) any one of five types of aberrant ratios can be found, and these gave the clues needed to build a DNA crossover model. The aberrant types are as follows:

All the aberrant ratios need to be explained, but the key types that led to the heteroduplex model are the 5:3, 3:5, and 3:1:1:3 ratios because these contain nonidentical sister spores. (Recall that the postmeiotic round of mitosis is expected to produce identical sister genotypes.) Nonidentical sister spore genotypes must reflect heteroduplex DNA in the meiotic product that gave rise to the pair, that is, DNA with a segment in which one strand is the nucleotide sequence of the A allele and one strand is the nucleotide sequence of the a allele. Upon mitosis the two sister cells resulting from division of such a heteroduplex-containing nucleus will be different, one A and one a. This process is shown in the following sketch; A and a represent the site in the gene at which the alleles differ:

Figure 5-18 shows one model of how heteroduplexes might form, the so-called Meselson-Radding model. In the following discussion it is important to refer closely to this figure. A strand from the DNA molecule of one chromatid (blue) breaks, unwinds, and "invades" a nonsister chromatid (yellow). New strand synthesis and joining of broken ends results in a peculiar type of half-helix crossover called a Holliday junction. One of the immediate products is a blue/ yellow heteroduplex region. If the Holliday junction migrates by directional unwinding and rewinding of the helix in one direction, then regions are created in which there are two aligned blue/yellow heteroduplexes (shown to the right in the figure). It has been shown that a Holliday junction can resolve itself in two different ways, involving either a "horizontal" breakage and reunion of the inner two strands at the junction, or a "vertical" breakage and reunion of the outer two strands at the junction. The vertical type produces "genuine" crossover chromatidsthat is, whole DNA double-helix crossovers. The horizontal resolution results in a noncrossover conformation. Hence the model suggests that the heteroduplex can be an essential precursor of the genetically detectable chromatid crossover.

If there is no migration of the junction, the resulting octad would be a 5:3 (don't forget there are two other outer chromatids not shown in the diagram). However, a heteroduplex is an unstable structure involving a mismatch of DNAperhaps a G·T pairand enzymes are known that recognize such mismatches and cut out one of the mispaired strands and replace it with one containing a properly hydrogen-bonded nucleotide (Figure 5-19). This type of mismatch repair of the unmigrated structure would either give a 6:2 ratio or flip back to a normal 4:4. In the heteroduplexes resulting from migration, repair can produce any of the other aberrant ratios. In these regions if no repair occurs, the 3:1:1:3 ratio results.

The Meselson-Radding model is only one of a number of models based on heteroduplex formation. The recombination mechanism in this model is initiated by a single-strand break in DNA. In another model, the recombination mechanism is initiated by a double-strand break, and there is support for this model from studies on yeast recombination. However, there is no model that is generally agreed to be applicable to all eukaryotes.

MESSAGE
The observation of rare cases of nonidentical ascospore pairs in fungal octads suggests that DNA heteroduplexes can form that are the basis of crossovers. The extent of the heteroduplex region and the repair of DNA mismatches in the heteroduplexes can explain all the aberrant ratios observed.

© 1999 by W. H. Freeman and Company. All rights reserved.