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复制叉上的ATM和ATR激酶信号。ATM激酶是由DNA双链断裂激活的，而ATR激酶是由单链（ssDNA）断裂诱导的。然而，通路之间存在串扰，ATM和ATR磷酸化一组重叠的底物。为了确定ATM激酶信号通路，我们使用了ATM激酶抑制剂KU55933和KU60019来短暂抑制细胞中的ATM激酶活性。使用这种创新的方法，我们表明急性ATM激酶抑制和ATM蛋白破坏的后果是不同的。在这里，我们表明急性ATM激酶抑制阻止DNA合成。这是令人惊讶的，因为不表达ATM蛋白的辐照细胞由于抑制晚起源放电的缺陷而不能阻止DNA合成。链伸长阻滞对s期内检查点的贡献很难确定，因为诱导检查点的病变也直接阻滞复制分叉。虽然最近的证据表明ATR信号可以阻止链延伸，但ATM的作用尚未得到解决。我们假设ATM激酶的活性促进了复制分叉的稳定进展，并减弱了ATR激酶的活性。我们提出，急性ATM激酶抑制会阻碍受损复制叉的修复，导致ssDNA间隙的积累，从而诱导ATR激酶信号传导和s期内检查点。这挑战了ATM激酶破坏使s期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激酶信号传导的尖锐工具，使用基于单DNA纤维的技术，可以可视化多个起源和从这些起源产生的单个复制分叉。我们将首次研究急性ATM激酶抑制和ATM蛋白破坏后细胞的起源密度和复制叉速度。

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.