Laboratory of Protein Structure and Function, Institute of Medicine and Pharmacy, Qiqihar Medical University, 161006, Qiqihar, Heilongjiang, China.
Division of Oncology Research and Schulze Center for Novel Therapeutics, Mayo Clinic College of Medicine, 55905, Rochester, MN, USA.
Mol Biol Rep. 2022 Sep;49(9):8349-8357. doi: 10.1007/s11033-022-07650-4. Epub 2022 Jun 22.
WASHC1 is a member of the Wiskott-Aldrich syndrome protein (WASP) family and is involved in endosomal protein sorting and trafficking through the generation of filamentous actin (F-actin) via activation of the Arp2/3 complex. There is increasing evidence that WASHC1 is present in the nucleus and nuclear WASHC1 plays important roles in regulating gene transcription, DNA repair as well as maintaining nuclear organization. However, the multi-faceted functions of nuclear WASHC1 still need to be clarified.
We show here that WASHC1 interacts with several components of the minichromosome maintenance (MCM) 2-7 complex by using co-immunoprecipitation and in situ proximity ligation assay. WASHC1-depleted cells display normal DNA replication and S-phase progression. However, loss of WASHC1 sensitizes HeLa cells to DNA replication inhibitor hydroxyurea (HU) and increases chromosome instability of HeLa and 3T3 cells under condition of HU-induced replication stress. Re-expression of nuclear WASHC1 in WASHC1 3T3 cells rescues the deficiency of WASHC1 cells in the chromosomal stability after HU treatment. Moreover, chromatin immunoprecipitation assay indicates that WASHC1 associates with DNA replication origins, and knockdown of WASHC1 inhibits MCM protein loading at origins.
Since efficient loading of excess MCM2-7 complexes is required for cells to survive replicative stress, these results demonstrate that WASHC1 promotes cell survival and maintain chromosomal stability under replication stress through recruitment of excess MCM complex to origins.
WASHC1 是 Wiskott-Aldrich 综合征蛋白（WASP）家族的成员，通过激活 Arp2/3 复合物生成丝状肌动蛋白（F-actin），参与内体蛋白分选和运输。越来越多的证据表明，WASHC1 存在于细胞核中，核 WASHC1 在调节基因转录、DNA 修复以及维持核组织方面发挥着重要作用。然而，核 WASHC1 的多方面功能仍有待阐明。
我们通过免疫共沉淀和原位邻近连接分析显示，WASHC1 与几个微小染色体维持（MCM）2-7 复合物的成分相互作用。WASHC1 耗尽的细胞显示出正常的 DNA 复制和 S 期进程。然而，WASHC1 的缺失使 HeLa 细胞对 DNA 复制抑制剂羟基脲（HU）敏感，并在 HU 诱导的复制应激下增加 HeLa 和 3T3 细胞的染色体不稳定性。在 WASHC1 3T3 细胞中重新表达核 WASHC1 可挽救 WASHC1 缺失后 HU 处理后染色体稳定性的缺陷。此外，染色质免疫沉淀分析表明，WASHC1 与 DNA 复制起始点相关联，并且 WASHC1 的敲低抑制了起始点处的 MCM 蛋白加载。
由于大量 MCM2-7 复合物的有效加载对于细胞在复制应激下的存活是必需的，这些结果表明，WASHC1 通过募集过量的 MCM 复合物到起始点，促进细胞存活并维持染色体稳定性在复制应激下。
