Roles of fork protection complex and cdc7 kinase in fork-stabilization and replication checkpoint

DNA 复制、检查点激活和复制后染色单体内聚力的协调执行与复制叉机制密切相关。在本研究中，我们重点研究了复制复合体成分的人类同源物AND-1、Tim和Tipin，并确定了它们在S期的功能如下： 1．人类 Tim 和 Tipin 在细胞周期中形成复合物。免疫共沉淀实验表明，Tim 还与 Mcm2 相互作用，AND-1 与粘连蛋白 Smc1、Smc3 Rad21 和 Mcm7.2 相互作用。 AND-1、Tim 和 Tipin 位于复制焦点附近，是有效 DNA 复制所必需的。3． AND-1的耗竭会导致DNA损伤和细胞死亡。4． AND-1、Tim 和 Tipin 是有效复制检查点和促进抗紫外线 DNA 复制所必需的。 Tim 和 Tipin 也是复制应激中 Claspin 染色质负载所必需的。5。 AND-1 通过复制停滞而被磷酸化，复制停滞取决于 ATM/ATR 和 Cdc7 激酶。6． AND-1是高效重组修复所必需的。基于这些数据，我们提出AND-1、Tim和Tipin与粘连蛋白和复制因子形成复合物，协调S期的多个细胞事件，如DNA复制、检查点激活、姐妹染色单体凝聚和修复，从而在维持基因组完整性中发挥关键作用。

Coordinated execution of DNA replication, checkpoint activation and post-replicative chromatid cohesion is intimately related to the replication fork machinery. In this study, we focused on the human homologs of replication complex components, AND-1, Tim, and Tipin and identified their functions in S phase as below.1. Human Tim and Tipin form complex during cell cycle. Coimmunoprecipitation experiments reveals that Tim also interacts with Mcm2 and AND-1 interacts with cohesin Smc1, Smc3 Rad21 and Mcm7.2. AND-1, Tim and Tipin localize adjacent to replication foci and are required for efficient DNA replication.3. Depletion of AND-1 causes DNA damage and cell death.4. AND-1, Tim and Tipin are required for efficient replication checkpoint and promote UV-resistant DNA replication. Tim and Tipin are also required for chromatin loading of Claspin in replication stress.5. AND-1 is phosphorylated by replication arrest which depends on ATM/ATR and Cdc7 kinase.6. AND-1 is required for efficient recombinational repair.Based on these data, we propose that AND- 1, Tim and Tipin forms complexes with cohesin and replication factors and coordinates multiple cellular events in S phase, such as DNA replication, checkpoint activation, sister chromatid cohesion and repair, thus playing a pivotal role in maintenance of genome integrity.