Figure 5–52 Experiment demonstrating the rapid localization of repair proteins to DNA double-strand breaks. Human fibroblasts were x-irradiated to produce DNA double-strand breaks. Before the x-rays struck the cells, they were passed through a microscopic grid with x-ray-absorbing “bars” spaced 1 μm apart. This produced a striped pattern of DNA damage, allowing a comparison of damaged and undamaged DNA in the same nucleus. (A) Total DNA in a fibroblast nucleus stained with the dye DAPI. (B) Sites of new DNA synthesis due to repair of DNA damage, indicated by incorporation of BudR (a thymidine analog) and subsequent staining with fluorescently labeled antibodies to BudR (green).
(C) Localization of the Mre11 complex to damaged DNA as visualized by antibodies against the Mre11 subunit (red). Mre11 is a nuclease that processes damaged DNA in preparation for homologous recombination (see Figure 5–48). (A), (B), and (C) were processed 30 minutes after x-irradiation. (From B.E. Nelms et al., Science 280:590– 592, 1998. With permission from AAAS.)
Figure 5–53 Chromosome crossing-over occurs in meiosis. Meiosis is the process by which a diploid cell gives rise to four haploid germ cells, as described in detail in Chapter 17. Meiosis produces germ cells in which the paternal and maternal genetic information (red and blue) has been reassorted through chromosome crossovers. In addition, many short regions of gene conversion occur, as indicated.
Figure 5–54 Homologous recombination during meiosis can generate chromosome crossovers. Once the meiosis-specific protein Spo11 and the Mre11 complex break the duplex DNA and process the ends, homologous recombination can proceed along alternative pathways. One (right side of figure) closely resembles the double-strand break repair reaction shown in Figure 5–48 and results in chromosomes that have been “repaired” but have not crossed over. The other (left side with strand breaks as shown by the blue arrows) proceeds through a double Holliday junction and produces two chromosomes that have crossed over. During meiosis, homologous recombination takes place between maternal and paternal chromosome homologs when they are held tightly together (see Figure 17–54).
DNA (see Figure 5–21). A specialized nuclease then rapidly degrades the ends bound by Spo11, removing the protein along with the DNA and leaving protruding 3ʹ single-strand ends.
At this point, many of the recombination reactions resemble those described above for the repair of double-strand breaks; indeed, some of the same proteins are used for both processes. However, several meiosis-specific proteins direct them to perform their tasks somewhat differently, resulting in the distinctive outcomes observed for meiosis. Another important difference is that, in meiosis, recombination occurs preferentially between maternal and paternal chromosomal homo-logs rather than between the newly replicated, identical DNA duplexes that pair in double-strand break repair. In the sections that follow, we describe in more detail those aspects of homologous recombination that are especially important for meiosis.
Of special importance in meiosis is an intermediate known as a Holliday junction or cross-strand exchange (Figure 5–55). Each Holliday junction can adopt multiple conformations and a special set of recombination proteins binds to, and thereby stabilizes, the open, symmetric isomer.