Data_Sheet_1_Uncoupling of DNA Replication and Centrosome Duplication Cycles Is a Primary Cause of Haploid Instability in Mammalian Somatic Cells.ZIP

Mammalian haploid somatic cells are unstable and prone to diploidize, but the cause of haploid instability remains largely unknown. Previously, we found that mammalian haploid somatic cells suffer chronic centrosome loss stemming from the uncoupling of DNA replication and centrosome duplication cycles. However, the lack of methodology to restore the coupling between DNA replication and centrosome duplication has precluded us from investigating the potential contribution of the haploidy-linked centrosome loss to haploid instability. In this study, we developed an experimental method that allows the re-coupling of DNA and centrosome cycles through the chronic extension of the G1/S phase without compromising cell proliferation using thymidine treatment/release cycles. Chronic extension of G1/S restored normal mitotic centrosome number and mitotic control, substantially improving the stability of the haploid state in HAP1 cells. Stabilization of the haploid state was compromised when cdk2 was inhibited during the extended G1/S, or when early G1 was chronically extended instead of G1/S, showing that the coupling of DNA and centrosome cycles rather than a general extension of the cell cycle is required for haploid stability. Our data indicate the chronic centriole loss arising from the uncoupling of centrosome and DNA cycles as a direct cause of genome instability in haploid somatic cells, and also demonstrate the feasibility of modulation of haploid stability through artificial coordination between DNA and centrosome cycles in mammalian somatic cells.

哺乳动物单倍体体细胞（mammalian haploid somatic cells）稳定性较差，且极易发生二倍体化，但其单倍体不稳定的具体成因迄今仍未明确。此前我们发现，哺乳动物单倍体体细胞存在慢性中心体（centrosome）缺失现象，该现象源于DNA复制与中心体复制周期的解偶联。然而，由于缺乏能够恢复DNA复制与中心体复制周期偶联的实验方法，我们无法探究单倍体相关的中心体缺失对单倍体不稳定性的潜在贡献。本研究中，我们开发了一种实验方法：通过胸腺嘧啶（thymidine）处理/释放循环，在不影响细胞增殖的前提下慢性延长G1/S期，从而实现DNA与中心体周期的重新偶联。G1/S期的慢性延长恢复了正常的有丝分裂中心体数目与有丝分裂调控功能，显著提升了HAP1细胞中单倍体状态的稳定性。若在延长的G1/S期内抑制细胞周期蛋白依赖性激酶2（cdk2），或是将细胞周期慢性阻滞于早期G1期而非G1/S期，则单倍体状态的稳定效应会被破坏，这表明实现单倍体稳定性所需的是DNA与中心体周期的偶联，而非细胞周期的一般性延长。我们的研究数据表明，中心体与DNA周期解偶联所引发的慢性中心粒（centriole）缺失，是哺乳动物单倍体体细胞基因组不稳定的直接诱因；同时也证实了通过人工协调DNA与中心体周期来调控哺乳动物体细胞单倍体稳定性的可行性。