Crossover Fixation Could Maintain Identical Repeats

KEY CONCEPTS
-Unequal crossing-over changes the size of a cluster of tandem repeats.
-Individual repeating units can be eliminated or can spread through the cluster.
Not all duplicated copies of genes become pseudogenes. How can selection prevent the accumulation of deleterious mutations?
The duplication of a gene is likely to result in an immediate relaxation of the selection pressure on the sequence of one of the two copies. Now that there are two identical copies, a change in the sequence of one will not deprive the organism of a functional product, because the original product can continue to be encoded by the other copy. Then, the selective pressure on the two genes is diffused until one of them mutates sufficiently away from its original function to refocus all the selective pressure on the other.
Immediately following a gene duplication, changes might accumulate more rapidly in one of the copies, eventually leading to a new function (or to its disuse in the form of a pseudogene). If a new function develops, the gene then evolves at the same, slower rate characteristic of the original function. Probably this is the sort of mechanism responsible for the separation of functions between embryonic and adult globin genes.
Yet, there are instances in which duplicated genes retain the same function, encoding identical or nearly identical products. Identical polypeptides are encoded by the two human α-globin genes, and there is only a single amino acid difference between the two γ-globin polypeptides. How does selection maintain their sequence identities?
The most obvious possibility is that the two genes do not actually have identical functions but instead differ in some (undetected) property, such as time or place of expression. Another possibility is that the need for two copies is quantitative because neither by itself produces a sufficient amount of product.
However, in more extreme cases of repetition, it is impossible to avoid the conclusion that no single copy of the gene is essential. When there are many copies of a gene, the immediate effects of mutation in any one copy must be very slight. The consequences of an individual mutation are diluted by the large number of copies of the gene that retain the wild-type sequence.

Many mutant copies could accumulate before a lethal effect is generated. Lethality becomes quantitative, a conclusion reinforced by the observation that half of the units of the rDNA cluster of X. laevis or D. melanogaster can be deleted without ill effect. So how are these units prevented from gradually accumulating deleterious mutations? What chance is there for the rare favorable mutation to display its advantages in the cluster?
The basic principle of hypotheses that explain the maintenance of identity among repeated copies is to suppose that nonallelic genes are continually regenerated from one of the copies of a preceding generation. In the simplest case of two identical genes, when a mutation occurs in one copy, either it is by chance eliminated (because the sequence of the other copy takes over) or it is spread to both duplicates. Spreading exposes a mutation to selection. The result is that the two genes evolve together as though only a single locus existed. This is called concerted evolution or coincidental evolution. It can be applied to a pair of identical genes or (with further assumptions) to a cluster containing many genes. For example, the tandemly repeated rRNA gene copies discussed extensively earlier in the chapter show concerted evolution. rDNA clusters tend to have identical copies within genomes of a wide variety of prokaryotic and eukaryotic organisms, while showing variation among different species.
One mechanism for this concerted evolution is that the sequences of the nonallelic genes are directly compared with one another and homogenized by enzymes that recognize any differences. This can be done by exchanging single strands between them to form a duplex in which one strand derives from one copy and one strand derives from the other copy. Any differences are revealed as improperly paired bases, which are recognized by enzymes able to excise and replace a base, so that only A-T and G-C pairs remain.
This type of event is called gene conversion and is associated with genetic recombination. Researchers should be able to ascertain the scope of such events by comparing the sequences of duplicate genes. If these duplicate genes are subject to concerted evolution, we should not see the accumulation of synonymous substitutions  between them because the homogenization process applies to these as well as to the nonsynonymous substitutions (those that do change the amino acid sequence). We know that the extent of the maintenance mechanism need not extend beyond the gene itself because there are cases of duplicate genes whose flanking sequences are entirely different. Indeed, we might see abrupt boundaries that mark the ends of the sequences that were homogenized.
We must remember that the existence of such mechanisms can invalidate the determination of the history of such genes via their divergence, because the divergence reflects only the time since the last homogenization/regeneration event, not the original duplication.
The crossover fixation model suggests that an entire cluster is subject to continual rearrangement by the mechanism of unequal crossing-over. Such events can explain the concerted evolution of multiple genes if unequal crossing-over causes all the copies to be physically regenerated from one copy.
Following the sort of event , for example, the chromosome carrying a triple locus could suffer deletion of one of the genes. Of the two remaining genes, 1.5 represent the sequence of one of the original copies; only a half of the sequence of the other original copy has survived. Any mutation in the first region now exists in both genes and is subject to selection.

Tandem clustering provides frequent opportunities for “mispairing” of loci whose sequences are the same, but that lie in different positions in their clusters. By continually expanding and contracting the number of units via unequal crossing-over, it is possible for all the units in one cluster to be derived from rather a small proportion of those in an ancestral cluster. The variable lengths of the spacers are consistent with the idea that unequal crossing-over events take place in spacers that are internally mispaired. This can explain the homogeneity of the genes compared with the variability of the spacers. The genes are exposed to selection when individual repeating units are amplified within the cluster; however, the spacers are functionally irrelevant and can accumulate changes.
In a region of nonrepetitive DNA, recombination occurs between precisely matching points on the two homologous chromosomes, thus generating reciprocal recombinants. The basis for this
precision is the ability of two duplex DNA sequences to align exactly. We know that unequal recombination can occur when there are multiple copies of genes whose exons are related, even though their flanking and intervening sequences might differ. This happens because of the mispairing between corresponding exons in nonallelic genes.
Imagine how much more frequently misalignment must occur in a tandem cluster of identical or nearly identical repeats. Except at the very ends of the cluster, the close relationship between successive repeats makes it impossible even to define the exactly corresponding repeats! This has two consequences: There is continual adjustment of the size of the cluster; and there is
homogenization of the repeating unit. Consider a sequence consisting of a repeating unit “ab” with ends “x” and “y.” If we represent one chromosome in black and the other in red, the exact alignment between “allelic” sequences would be as follows:
xababababababababababababababababy
xababababababababababababababababy
It is likely, however, that any sequence ab in one chromosome could pair with any sequence ab in the other chromosome. In a misalignment such as
xababababababababababababababababy
xababababababababababababababababy
the region of pairing is no less stable than in the perfectly aligned pair, although it is shorter. Researchers do not know very much about how pairing is initiated prior to recombination, but very likely it begins between short, corresponding regions and then spreads.
If it begins within highly repetitive satellite DNA, it is more likely than not to involve repeating units that do not have exactly corresponding locations in their clusters.
Now suppose that a recombination event occurs within the unevenly paired region. The recombinants will have different numbers of repeating units. In one case, the cluster has become longer; in the other, it has become shorter,
xababababababababababababababababy
×
xababababababababababababababababy
↓
xababababababababababababababababababy
+
xababababababababababababababy
where “×” indicates the site of the crossover.
If this type of event is common, clusters of tandem repeats will undergo continual expansion and contraction. This can cause a particular repeating unit to spread through the cluster, as illustrated in FIGURE 1. Suppose that the cluster consists initially of a sequence abcde, where each letter represents a repeating unit.
The different repeating units are related closely enough to one another to mispair for recombination. Then, by a series of unequal recombination events, the size of the repetitive region increases or decreases, and one unit spreads to replace all the others.

FIGURE 1 Unequal recombination allows one particular repeating unit to occupy the entire cluster. The numbers indicate the length of the repeating unit at each stage.
The crossover fixation model predicts that any sequence of DNA that is not under selective pressure will be taken over by a series of identical tandem repeats generated in this way. The critical assumption is that the process of crossover fixation is fairly rapid relative to mutation so that new mutations either are eliminated (their repeats are lost) or come to take over the entire cluster. In the case of the rDNA cluster, of course, a further factor is imposed by selection for a functional transcribed sequence.