National Centre for Cell Science, NCCS Complex, Ganeshkhind Road, Pune, Maharashtra 411007, India.
Department of Biotechnology, Savitribai Phule Pune University, Ganeshkhind Road, Pune, Maharashtra 411007, India.
J Biol Chem. 2019 Oct 11;294(41):14879-14895. doi: 10.1074/jbc.RA118.007055. Epub 2019 Aug 14.
F-box protein 31 (FBXO31) is a reported putative tumor suppressor, and its inactivation due to loss of heterozygosity is associated with cancers of different origins. An emerging body of literature has documented FBXO31's role in preserving genome integrity following DNA damage and in the cell cycle. However, knowledge regarding the role of FBXO31 during normal cell-cycle progression is restricted to its functions during the G/M phase. Interestingly, FBXO31 levels remain high even during the early G phase, a crucial stage for preparing the cells for DNA replication. Therefore, we sought to investigate the functions of FBXO31 during the G phase of the cell cycle. Here, using flow cytometric, biochemical, and immunofluorescence techniques, we show that FBXO31 is essential for maintaining optimum expression of the cell-cycle protein cyclin A for efficient cell-cycle progression. Stable FBXO31 knockdown led to atypical accumulation of cyclin A during the G phase, driving premature DNA replication and compromised loading of the minichromosome maintenance complex, resulting in replication from fewer origins and DNA double-strand breaks. Because of these inherent defects in replication, FBXO31-knockdown cells were hypersensitive to replication stress-inducing agents and displayed pronounced genomic instability. Upon entering mitosis, the cells defective in DNA replication exhibited a delay in the prometaphase-to-metaphase transition and anaphase defects such as lagging and bridging chromosomes. In conclusion, our findings establish that FBXO31 plays a pivotal role in preserving genomic integrity by maintaining low cyclin A levels during the G phase for faithful genome duplication and segregation.
F-box 蛋白 31（FBXO31）是一种报道的潜在肿瘤抑制因子，其由于杂合性丢失而失活与不同起源的癌症有关。越来越多的文献记录了 FBXO31 在 DNA 损伤后和细胞周期中维持基因组完整性的作用。然而，关于 FBXO31 在正常细胞周期进展中的作用的知识仅限于其在 G/M 期的功能。有趣的是，即使在 G1 早期，FBXO31 水平仍然很高，这是为 DNA 复制准备细胞的关键阶段。因此，我们试图研究 FBXO31 在细胞周期的 G 期的功能。在这里，我们使用流式细胞术、生化和免疫荧光技术，表明 FBXO31 对于维持细胞周期蛋白 A 的最佳表达以促进细胞周期进程是必需的。稳定的 FBXO31 敲低导致 G 期 cyclin A 的异常积累，导致过早的 DNA 复制和微染色体维持复合物的加载受损，导致从较少的起始点进行复制和 DNA 双链断裂。由于这些固有的复制缺陷，FBXO31 敲低的细胞对复制应激诱导剂高度敏感，并表现出明显的基因组不稳定性。进入有丝分裂后，复制缺陷的细胞在前期到中期的转变中出现延迟，以及后期缺陷，如滞后和桥接染色体。总之，我们的发现表明，FBXO31 通过在 G 期维持低 cyclin A 水平来维持忠实的基因组复制和分离，从而在维持基因组完整性方面发挥关键作用。
