Regulation of DNA replication fork progression by ATM kinase activity

描述(由申请人提供)：本提案的长期目标是了解DNA复制分叉处的ATM和ATR激酶信号。ATM激酶是由DNA双链断裂激活的，ATR激酶是由单链(SsDNA)间隙诱导的。然而，两条通路之间存在串扰，ATM和ATR将一组重叠的底物磷酸化。为了确定不可或缺的ATM激酶信号，我们使用ATM激酶抑制剂KU55933和KU60019来瞬时抑制细胞中的ATM激酶活性。使用这种创新的方法，我们证明了急性ATM激酶抑制和ATM蛋白破坏的后果是不同的。在这里，我们展示了急性ATM激酶抑制阻止DNA合成。这是令人惊讶的，因为没有ATM蛋白表达的受辐射细胞不会因为抑制晚期起源激发的缺陷而阻止DNA合成。链延长抑制对S期内检查点的作用一直很难确定，因为导致检查点的损伤也直接阻止复制分叉。虽然最近的证据表明ATR信号可以阻止链的延长，但ATM的作用还没有被解决。我们假设ATM激酶活性促进复制分叉的稳定进展，并减弱ATR激酶活性。我们认为，急性ATM激酶抑制阻碍了受损复制分叉的修复，导致诱导ATR激酶信号转导和S阶段检查点的单链DNA缺口的积累。这挑战了ATM激酶中断使S阶段内检查点失效的范式。在这里，我们将结合使用KU55933和KU60019作为抑制ATM激酶信号的锐利工具和基于单DNA纤维的技术，该技术允许可视化多个起始点和从这些起始点出现的单个复制分叉。我们将在急性ATM激酶抑制和ATM蛋白中断后，进行细胞内起源密度和复制分叉速度的第一次调查。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand ATM and ATR kinase signaling at DNA replication forks. While ATM kinase is activated by DNA double strand breaks, ATR kinase is induced by single-stranded (ssDNA) gaps. However, crosstalk exists between the pathways and ATM and ATR phosphorylate an overlapping set of substrates. To identify indispensable ATM kinase signaling we used the ATM kinase inhibitors KU55933 and KU60019 to transiently inhibit ATM kinase activity in cells. Using this innovative approach we showed that the consequences of acute ATM kinase inhibition and ATM protein disruption are distinct. Here we show that acute ATM kinase inhibition arrests DNA synthesis. This is surprising since irradiated cells that express no ATM protein do not arrest DNA synthesis due to a defect in the inhibition of late origin firing. The contribution of chain elongation arrest to the intra-S-phase checkpoint has been difficult to establish since lesions that induce the checkpoint also directly arrest replication forks. While recent evidence indicates that ATR signaling can arrest chain elongation, the role of ATM has not been addressed. We hypothesize that ATM kinase activity promotes the steady progression of replication forks and attenuates ATR kinase activity. We propose that acute ATM kinase inhibition impedes the repair of damaged replication forks causing an accumulation of ssDNA gaps that induce ATR kinase signaling and the intra-S-phase checkpoint. This challenges the paradigm that ATM kinase disruption disables the intra-S-phase checkpoint. Here we will combine the use of KU55933 and KU60019 as sharp tools to inhibit ATM kinase signaling with single DNA fiber-based technology that allows the visualization of multiple origins and individual replication forks emerging from those origins. We will undertake the first investigation of origin density and replication fork velocity in cells following acute ATM kinase inhibition and ATM protein disruption.