Interaction of the Warsaw breakage syndrome DNA helicase DDX11 with the replication fork-protection factor Timeless promotes sister chromatid cohesion

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS.

姐妹染色单体黏连（sister chromatid cohesion）的建立与DNA复制（DNA replication）相偶联，但其潜在的分子机制尚未完全阐明。DDX11（亦称作ChlR1）是一类隶属于超家族2（super-family 2）的、含铁硫簇（Fe-S cluster）的DNA解旋酶（DNA helicase），与华沙断裂综合征（Warsaw breakage syndrome, WABS）密切相关。本研究中，我们探究了DDX11在人类细胞中姐妹染色单体黏连建立过程中的作用。我们证实，DDX11可通过保守肽基序（conserved peptide motif）与复制叉保护复合体（replication fork-protection complex）的组分之一Timeless发生相互作用。DDX11与Timeless的相互作用对于间期（interphase）与有丝分裂（mitosis）阶段的姐妹染色单体黏连至关重要。免疫荧光（immunofluorescence）实验进一步揭示，黏连蛋白（cohesin）与染色质（chromatin）的结合依赖于DDX11的存在。最后，我们通过SIRF分析证实DDX11定位于新生DNA（nascent DNA）区域。此外，我们发现DDX11可与Timeless协同促进黏连蛋白结合至DNA复制叉，且体外（in vitro）实验中重组纯化的黏连蛋白（recombinant purified cohesin）可与DDX11发生相互作用。综上，本研究结果明确了DDX11-Timeless相互作用在协调DNA复制与姐妹染色单体黏连过程中的关键作用，这为阐明华沙断裂综合征的分子基础提供了重要的理论参考。