Rad9/53BP1 controls the initiation and the elongation of DNA replication in budding yeast

In budding yeast, DNA lesions and stalled replication forks are sensed by the apical checkpoint kinase Mec1/ATR, which leads to the downstream activation of the effector kinase Rad53/CHK1. This activation depends on Rad9 and Mrc1, two checkpoint mediators that integrate the nature of the challenge in different phases of the cell cycle. Rad9 mediates the activation of the DNA damage checkpoint throughout the cell cycle, while the function of Mrc1 is restricted to the S phase of the cell cycle, when it travels with the replication fork and activates the DNA replication checkpoint in response to a variety of replication impediments. In this scenario, the role of Rad9 in S phase has been largely disregarded since the discovery of Mrc1, because Rad9 is dispensable for the timely activation of Rad53 in response to the drug hydroxyurea, which halts forks, and is only recruited to those stalled forks when Mrc1 is absent. Thus, Rad9 is simply believed to act as a backup pathway for Mrc1 during replication. We have re-evaluated the role of Rad9 when DNA damage arises during replication and characterized its functional interplay with Mrc1. To this end, we have used genome-wide approaches, single-molecule analysis, pulsed-field and 2D gel electrophoresis, as well as a careful combination of different replication-challenging drugs. We have found that both Mrc1 and Rad9 play distinct but complementary functions in the replication stress response during S phase, for they coordinate the early and late functions of Rad53, respectively. While Mrc1 is responsible for the fast activation of Rad53 in response to fork-halting drugs in order to repress late origins, Rad9 maintains Rad53 in an active state during prolonged fork arrest and is necessary to sustain this response for long periods. Remarkably, we also have found that Rad9 possesses the unprecedented activity of slowing down replication fork progression in response to DNA damage. This work thus restores the legitimate role of Rad9 as a central actor in the maintenance of genome integrity during replication. This has important implications for our understanding of the management of the checkpoint during perturbed replication in human cells, for Rad9 has three orthologues, 53BP1, BRCA1 and MDC1, whose contribution to this aspect of genome integrity remains largely unexplored.在酿酒酵母（budding yeast）中，DNA损伤（DNA lesions）与停滞的复制叉（stalled replication forks）可被顶端检查点激酶（apical checkpoint kinase）Mec1/ATR感知，进而激活下游效应激酶（effector kinase）Rad53/CHK1。该激活过程依赖于检查点介导因子（checkpoint mediators）Rad9与Mrc1，二者可整合细胞周期（cell cycle）不同阶段所面临的应激性质。Rad9介导全细胞周期中的DNA损伤检查点（DNA damage checkpoint）激活，而Mrc1的功能仅局限于S期（S phase）：此时它伴随复制叉（replication fork）移动，并针对各类复制障碍（replication impediments）激活DNA复制检查点（DNA replication checkpoint）。自Mrc1被发现以来，Rad9在S期的作用长期被忽视，这是因为在羟基脲（hydroxyurea，一种可使复制叉停滞的药物）处理下，Rad9对于Rad53的及时激活并非必需，且仅当Mrc1缺失时，Rad9才会被招募至停滞的复制叉。因此，Rad9曾被简单认为是Mrc1在复制过程中的后备通路。本研究重新评估了Rad9在复制过程中DNA损伤出现时的作用，并阐明了其与Mrc1的功能互作机制。为此，我们采用了全基因组方法（genome-wide approaches）、单分子分析（single-molecule analysis）、脉冲场及二维凝胶电泳（pulsed-field and 2D gel electrophoresis），以及多种复制应激药物（replication-challenging drugs）的组合处理方案。研究发现，Mrc1与Rad9在S期的复制应激响应（replication stress response）中发挥着既独立又互补的功能：二者分别协调Rad53的早期与晚期激活功能。其中，Mrc1负责响应复制叉停滞药物，快速激活Rad53以抑制晚期复制起始位点（late origins）；而Rad9则在复制叉长期停滞期间维持Rad53的激活状态，是长期维持该响应的必需因子。值得注意的是，我们还发现Rad9具备前所未有的活性：可响应DNA损伤而减缓复制叉的前进速度。综上，本研究恢复了Rad9作为复制过程中维持基因组完整性（genome integrity）核心因子的应有地位。鉴于Rad9的同源基因（orthologues）包括53BP1、BRCA1与MDC1，而目前学界对这些同源基因在基因组完整性维护该方面的贡献仍知之甚少，本研究对于我们理解人类细胞中应激复制过程中的检查点调控机制具有重要意义。