Department of Molecular Biology, CABIMER-CSIC, Seville, Spain.
PLoS One. 2010 Aug 12;5(8):e12143. doi: 10.1371/journal.pone.0012143.
The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Delta can be partially or totally suppressed if SWR1 is not formed (swr1Delta), if it forms but cannot bind to chromatin (swc2Delta) or if it binds to chromatin but lacks histone replacement activity (swc5Delta and the ATPase-dead swr1-K727G). These results suggest that in htz1Delta the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Delta, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Delta. In addition, SWR1 causes DSBs sensitivity in htz1Delta; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling.
SWR1 复合物在特定染色质区域用变体 H2A.Z（酵母中的 Htz1）替代经典组蛋白 H2A。核小体结构的这种动态改变为调节转录、基因沉默、染色体分离和 DNA 修复提供了分子机制。在这里，我们表明，如果 SWR1 没有形成（swr1Δ），如果它形成但不能结合到染色质上（swc2Δ），或者如果它结合到染色质上但缺乏组蛋白替换活性（swc5Δ 和 ATP 酶缺陷的 swr1-K727G），htz1Δ 中的遗传不稳定性、对不同细胞过程有损伤作用的药物的敏感性以及全基因组转录失调可以部分或完全被抑制。这些结果表明，在 htz1Δ 中，由于缺乏后者，SWR1 的核小体重塑活性试图用 Htz1 替代 H2A，从而影响染色质完整性。这将损害转录，并且直接或间接地影响其他细胞过程。具体来说，我们表明，在 htz1Δ 中，SWR1 复合物通过依赖于 Ser129 磷酸化的机制导致可重组 DNA 损伤的积累，该修饰是对 DNA 损伤的反应，这表明 SWR1 复合物损害了 htz1Δ 中自发 DNA 损伤的修复。此外，SWR1 导致 htz1Δ 中 DSB 敏感性；一致地，在缺乏 Htz1 的情况下，SWR1 复合物结合在内切核酸酶 HO 诱导的 MAT 位点附近的 DSB 处，损害 DSB 诱导的检查点激活。我们的结果支持用 Htz1 逐步替代 H2A 的机制，并表明对这种机制的严格控制对于调节染色质动力学以及防止不完全核小体重塑的有害后果都是必不可少的。
