Institute of Genetics and Molecular Medicine, The University of Edinburgh, Edinburgh EH4 2XR, UK.
Biochem Biophys Res Commun. 2011 Nov 4;414(4):820-5. doi: 10.1016/j.bbrc.2011.10.021. Epub 2011 Oct 12.
DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to facilitate repair, the bulk genome becomes rapidly compacted protecting cells from further damage.
DNA 被包装成更高阶的染色质纤维来进行保护，但这可能会阻碍核过程，如 DNA 修复。尽管已经对信号转导和修复 DNA 损伤所需的因素进行了大量研究，但尚不清楚是否存在全球染色质纤维结构的伴随变化。在人类细胞中，DNA 双链断裂 (DSB) 的形成引发信号级联反应，导致 H2AX 磷酸化 (γH2AX)、染色质相关蛋白的快速募集以及受损部位的后续修复。KAP1 是一种转录共抑制因子，在 HCT116 细胞中，我们发现化学物质或电离辐射形成 DSB 后，会出现一波主要依赖于 ATM 的 KAP1 磷酸化。KAP1 和磷酸化的 KAP1 都很容易从细胞中提取出来，这表明它们没有结构作用，γH2AX 存在于可溶性染色质中，表明损伤部位没有附着在潜在的结构基质上。在 DSB 形成后，我们没有发现染色质纤维对微球菌核酸酶消化的敏感性同时发生变化。因此，为了直接研究高阶染色质纤维结构，我们使用了一种基于蔗糖梯度离心的生物物理沉淀技术，比较了 DNA DSB 形成前后从细胞中分离出的染色质纤维的构象。在损伤后，我们发现全局染色质纤维的紧密化，伴随着快速连接组蛋白去磷酸化，这与纤维更规则地折叠一致，或者纤维变形通过连接组蛋白稳定。我们认为，在 DSB 形成后，尽管局部染色质展开有利于修复，但大部分基因组会迅速紧凑，从而保护细胞免受进一步损伤。
