Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA.
Carcinogenesis. 2010 Jun;31(6):994-1002. doi: 10.1093/carcin/bgq035. Epub 2010 Feb 3.
Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR.
未能重新激活停滞或崩溃的 DNA 复制叉是基因组不稳定的潜在来源。同源重组 (HR) 是修复因复制停滞而导致的 DNA 损伤的主要机制。单链 DNA（ssDNA）结合蛋白复制蛋白 A（RPA）在多种 DNA 代谢过程中发挥重要作用。然而，DNA 损伤时 RPA2 高度磷酸化的作用尚不清楚。在这里，我们发现 RPA2 高度磷酸化与 ssDNA 和重组酶蛋白 Rad51 结合，以响应羟基脲（HU）处理引起的复制停滞。此外，RPA2 高度磷酸化对于 HU 后 Rad51 募集和 HR 介导的修复至关重要。然而，RPA2 高度磷酸化对于电离辐射（IR）诱导的 Rad51 焦点形成和 I-Sce-I 内切酶刺激的 HR 既不是必需的。此外，我们表明，表达 RPA2 磷酸化缺陷突变体后，在 HU 处理后而非暴露于 IR 后会导致染色体畸变增加。最后，我们证明在面临复制应激时，RPA2 高度磷酸化的丧失会导致细胞活力丧失，而表达高度磷酸化缺陷型 RPA2 或野生型 RPA2 的细胞对 IR 的敏感性相似。因此，我们的数据表明，RPA2 高度磷酸化通过促进 HR 在 DNA 复制受阻后维持基因组稳定性和细胞存活中发挥关键作用。
