March 2006
Volume 5
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March 2006
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Modifying Chromatin to Protect the Genome
DSBs induce the rapid phosphorylation of the H2AX isoform of histone H2A to form γH2AX (Rogakou EP et al. J Biol Chem 273: 5858–68, 1998), which is thought to play an important role in break repair (Fernandez-Capetillo O et al. DNA Repair 3: 959–67, 2004). The study cited at the top of this article (Shroff R et al. Curr Biol 14, 1703–11, 2004) and a second study (Ünal E et al. Mol Cell 16: 991–1002, 2004) provide a picture of one way that γH2AX protects the genome from damage. Shroff R et al. used chromatin immunoprecipitation (ChIP) to probe γH2AX formation and recruitment of the repair protein Mre11p at a site in the budding yeast genome where breaks can be formed in a controlled manner. The relative contribution of the two yeast damage-response kinases, Tel1p (ATM homolog) and Mec1p (ATR homolog), to H2AX phosphorylation was also determined. A panel of mutants in DNA damage response/repair genes was used to show that both Tel1p and Mec1p phosphorylate H2AX. Mutants blocking steps further down the DNA response/repair pathway had no effect on γH2AX formation, confirming that γH2AX formation is part of the initial DNA damage response. In the G1 phase of the cell cycle, Tel1p was responsible for most γH2AX formation, a finding similar to those obtained in studies of mammalian cells. ChIP analysis showed that γH2AX and Mre11p occupy distinct regions around the induced DSB. Mre11p, like other repair proteins, bound to sites directly adjacent to the DSB (within 1–2 kb). Conversely, γH2AX was present in a 40–50 kb region surrounding the break site. γH2AX was most abundant in a 3–5 kb band on either side of the break, with significant levels up to 25 kb from the peak site. Remarkably, very little γH2AX was detected at sites within 1–2 kb of the DSB, although ChIP analysis showed that histones were still present in this interval (Figure 1). Figure 1. γH2AX recruits cohesin to damage sites. Top—experimental data showing the broad region where γH2AX (red) forms; in contrast, repair proteins (here Mre11p, blue) bind in a narrow region. Bottom—nucleosomes containing γH2AX (red) recruit cohesin complexes (green rings), which use the unbroken sister chromatid to “splint” broken ends together, while leaving the ends themselves free for repair. ChIP, chromatin immunoprecipitation; DSB, DNA double-strand break. The disparity between the location of repair proteins and of γH2AX indicates that γH2AX most likely does not directly recruit repair proteins to DNA damage sites. Instead, we suggested that this large region of γH2AX creates chromosome structural changes that promote damage repair. Ünal E et al. (Mol Cell 16: 991–1002, 2004) and Ström L et al. (Mol Cell 16: 1003–15, 2004) provide support for this idea by examining the distribution of cohesin around a DSB. Cohesin is normally loaded onto chromosomes during replication, and holds sister chromatids together until mitosis. These papers report that DSBs provoke post-replicative cohesin loading in a large region, and that this additional cohesin is important for efficient DSB repair. The distribution of cohesin closely resembles that of γH2AX, suggesting that γH2AX might play a role in this damage-induced cohesin loading. Ünal E et al. showed that this is, in fact, the case. Mutants unable to form γH2AX do not recruit cohesin to DSBs and, consequently, have defects in repairing gamma ray–induced chromosome breaks. These findings suggest a picture where γH2AX formation and the subsequent recruitment of cohesin stabilize broken chromosomes, using the unbroken sister chromosome as a splint to hold broken ends together while leaving the actual site of damage open for repair proteins (Figure 1). This helps ensure that DSB repair occurs efficiently and with fidelity, maintaining genome integrity in the face of DNA damage and avoiding the genome rearrangements associated with cancer. |
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respond to a double-strand break (DSB) in their DNA by phosphorylating chromatin
in a large region surrounding the break site. In post-replicative cells, this
modification promotes the de novo deposition of cohesin, a multiprotein
complex that is normally loaded onto chromosomes during replication. DSB-induced
cohesin loading is likely to tether break ends close to the sister chromatid,
facilitating repair and helping the cell to maintain genome integrity.