Institute for Cancer Studies, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom.
Mol Biol Cell. 2010 Mar 1;21(5):739-52. doi: 10.1091/mbc.e09-07-0618. Epub 2010 Jan 6.
H2AX phosphorylation at serine 139 (gammaH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that gammaH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing gammaH2AX are not committed to death. gammaH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain gammaH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing gammaH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of gammaH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the gammaH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.
H2AX 丝氨酸 139 位的磷酸化（γH2AX）是 DNA 损伤和 DNA 复制应激的敏感标志物。在这里，我们发现，在 Chk1 缺失的 HCT116 或 SW480 细胞中，复制抑制剂而非电离辐射能强烈增强 γH2AX 的形成。虽然在这些细胞中，H2AX 的磷酸化先于细胞凋亡的诱导，但我们的结果表明，含有 γH2AX 的细胞并不一定会走向死亡。这些细胞中的 γH2AX 焦点与 RPA 焦点大部分重合，其形成依赖于必需的复制解旋酶辅助因子 Cdc45，表明 H2AX 的磷酸化发生在停滞的叉位点。然而，从复制抑制剂中释放出来的 Chk1 缺失细胞保留了 γH2AX 焦点，似乎并没有恢复复制性 DNA 合成。在从胸苷抑制中释放后，只有少数含有 γH2AX 焦点的 Chk1 缺失细胞会掺入 BrdU，并且在掺入 BrdU 的细胞中，DNA 合成不会发生在 γH2AX 焦点处。此外，这些细胞中持续存在激活的 ATM 和 Chk2。我们提出，Chk1 缺失细胞中的 γH2AX 焦点可能代表持续的复制叉损伤或放弃的位点，这些位点无法恢复 DNA 合成，但在 Chk1 抑制的死亡途径中不起直接作用。
