Homologous Recombination Has Additional Functions in Eukaryotes
As we have just described, homologous recombination in bacteria is required to repair double-stranded breaks in DNA, to restart collapsed replication forks, and to allow a cell's chromosomal DNA to recombine with DNA that enters via phage infection or conjugation. Homologous recombination is also required for DNA repair and the restarting of collapsed replication forks in eukaryotic cells. This requirement is illustrated by the fact that cells with defects in the proteins that promote recombination are hypersensitive to DNA damaging agents, especially those that break DNA strands. Furthermore, animals carrying mutations that interfere with homologous recombination are predisposed to certain types of cancer.
However, as we will discuss below, homologous recombination plays important additional roles in eukaryotic organisms. Most importantly, homologous recombination is critical for meiosis. During mejosis, homologous recombination is required for proper chromosome pairing and, thus, for maintaining the integrity of the genome. This recombination also reshuffles genes between the parental chromosomes, ensuring variation in the sets of genes passed to the next generation.
Homologous Recombination Is Required for Chromosome Segregation during Meiosis
As we saw in Chapter 7, meiosis involves two rounds of nuclear division, resulting in a reduction of the UNA content from the normal content of diploid cells (2N), to the content present in gametes (IN). Figure 10-14 shows schematically how the chromosomes are configured during these two division cycles. Before division, the cell has two copies of each chromosome (the homologs), one each that was inherited from its two parents, During S phase, these chromosomes are replicated to give a total DNA content of 4N. The products of replication—that is the sister chromatids—stay together. Then, in preparation for the first nuclear division, these duplicated homologous chromosomes must pair and align at the center of the cell. It is this pairing of homologs that requires homologous recombination (Figure 10-14). These events are care hilly timed. Recombination must be complete before the first nuclear division to allow the homologs to properly align and then separate. During this process, sister chromatids remain paired (see Chapter 7, Figure 7-16). Then, in the second nuclear division, it is the sister chromatids that separate. The products of this division are the four gametes, each with one copy of each chromosome (that is, the IN DNA content).
Without recombination, chromosomes often Tail to align properly for the first meiotic division, and, as a result, there is a high incidence of chromosome loss. This improper segregation of chromosomes, called nondisjunction, leads to a large number of gametes without the correct chromosome complement. Gametes with either too few or too many chromosomes cannot develop properly once fertilized; thus, a Failure in homologous recombination is often reflected in poor fertility. The homologous recombination events that occur during meiosis are called meiotic recombination.
Meiotic recombination also frequently gives rise to crossing over between genes on the two homologous parental chromosomes. This genetic exchange can be observed cytologically (Figure 10-15, top panel). An important consequence is that the alleles present on the parental DNA molecules are reasserted for the next generation.
Programmed Generation of Double-Stranded DNA Breaks Occurs during Meiosis
The developmental program needed for cells to successfully complete meiosis involves turning on the expression of many genes that are not needed during normal growth. One of these is SP011, This gene encodes a protein that introduces double-strand breaks in chromosomal DNA to initiate meiotic recombination.
The Spoil protein cuts the DNA at many chromosomal locations, with little sequence selectivity, but at a very specific time during meiosis. Spoil-mediated DNA cleavage occurs right around the time when the replicated homologous chromosomes start to pair. Spoil cut-sites, although frequent, are not randomly distributed along the DNA, Rather, the cut-sites ate located most commonly in chromosomal regions that are not tightly packed with nucleosomes, such as promoters controlling gene transcription (see Chapters 7 and 17). Regions of DNA that experience a high frequency of DSBs also show a high frequency of recombination, Thus, the most commonly used Spoil DNA cleavage sites, like chi sites, are hotspots for recombination.
FIGURE TO-14 DNfl dynamics during meiosis. Here, only one type of chromosome is shown for clanty. The two homology are shown, in red and blue, after thf?y have been duplicated by a round of DNA replication. Homologous recombination is required to pit these homologous chromosomes in preparation (or the first nuclear division, rhis recombination can also lead to crossing over, as is shown here between the A and B genes.
FIGURE 10-15 Cytologfcal view of crossing over. Reaprocal crossing over directly visualized in hamster cells In tissue culture. Chromosomes whose DNA contains bromodeoxyundme in place of thymidine in both strands appear light after treatment with Giemse stain, whereas those containing DMA substituted in only one strand appear dark. After two generations of growth in bromo-deoxyuridine, one newly replicated chomatid has only one of its strands substituted, whereas its sister has both substituted. Thus, sister chromatids can be distinguished by staining. Then crossovers are easily detected as alternating lengths of light and darf; (top). Similar recombinant chromosomes are also seen when mitobcally growing cells are treated with a DNA-damagrng agent (bottom). (Source: tour teSy o( Sheldon Wolff and Jody BodyCote)
The mechanism of DNA cleavage is as follows. A specific tyrosine side chain in the Spoil protein attacks the phosphodiester backbone to cut the DNA and genernto a cava lent complex between the protein and the severed DNA strand (Figure 10-16). Two subunits of Spoil cleave the DNA two nucleotides apart on the two DNA si rands to make a staggered double-strand break. Spoil shares this DNA cleavage mechanism with the DNA topoisomerases and the site-specific recombinases (see Chapter 6 and Chapter 11). in fact, Spoil appears to be a distant cousin of these ennymes.
The fact that Spoil cleavage involves a covalent protein-DNA complex has two consequences. First, the 5' ends of the DNA at the site of Spoil cleavage are covalently bound to ¡he enzyme. It is these Spol 1-linked 5r DNA ends that are the initial sites of DNA processing to create the ssDNA tails required for assembly of RecA-like proteins and initiation of DNA strand invasion (see below). Second, the energy of the cleaved DNA phosphodiester bond is stored in the bound protein-DNA linkage, and so the DNA strands ran be resealed by a simple reversal of the cleavage reaction (see Chapter 11, Figure 11-7). This reselling can occur when cells receive a signal to stop proceeding with meiosis.
FIGURE 10-16 Mechanism of cleavage by Spot 1. The OH group of a tyrosine in the Spol 1 protein attacks the DNA to form a rovatent protein-DMA linkage. Two subur.itsof Spo 11 are required to generate a double-strartded DMA break, one to attack each of the two DMA strands. Note, because of this cleavage rr.ech&nism, the DSE can be reseated by the simple reversal of the cleavage reaction.
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