DNA damage signaling to dormant origins of replication

高等真核生物进化的机制是在多个来源启动DNA复制 染色体。在~50,000个复制子中的每个复制子中，在单个起始处激活复制解旋酶就足够了 在没有压力的情况下复制人类基因组。这~50,000个原产地是从五到五个 超过许可原产地的十倍。在起始处激活额外的复制解旋酶，否则 应激后观察到被动复制。这种原始使用的可塑性是一种简单的恢复机制 停滞和折叠的复制叉之间的DNA复制。将发射原点限制为一次的机制(S) 每个复制子在没有压力的情况下是未知的。我们最近报道了DNA损伤信号转导通路 ATR和Chk1在没有应激的情况下抑制复制解旋酶的激活。在初步研究中，我们 表明Chk1的活性与Chk1上的ATR依赖的磷酸化密切相关。 它们在细胞中的半衰期惊人地短。我们认为，这是一个高度创新的机制 这将Chk1激酶的活性定位在ATR活性复制解旋酶的附近。我们还展示了 Rif1是磷酸化的，以前曾被牵连到复制时间的调节中，并且 磷酸化的Rif1与蛋白磷酸酶1α(pp1α)结合。基于这些发现，我们假设 Chk1激酶活性在活性复制解旋酶周围产生一环Rif1-pp1α，这限制了cdc7 在复制子上激活依赖于激酶的起始点。在目标1中，我们将研究一种新的机械论范式 在没有压力的情况下，将DNA损伤定位到小体积的细胞核。在《目标2》中我们将 研究限制复制子上复制解旋酶激活的分子机制 没有压力。在目标3中，我们将研究ATR激酶抑制和适形辐射对脑细胞的影响。 荷瘤小鼠的免疫反应。这些研究的影响力很大，因为它们将确定一个基本的 决定种间距离、基因组稳定性和细胞分裂率的机制 真核生物。由于这种机制在T细胞中可能会减弱，这些研究将提供基本的见解 转化为适应性免疫反应。我们的研究可能会产生立竿见影的影响，因为ATR和Chk1激酶 这里使用的抑制剂是临床试验。

Higher eukaryotes evolved with mechanisms that initiate DNA replication at multiple origins on multiple chromosomes. Activation of the replicative helicase at a single origin in each of ~50,000 replicons is sufficient to replicate the human genome in the absence of stress. These ~50,000 origins are selected from a five- to tenfold excess of licensed origins. Activation of additional replicative helicases at origins that would otherwise be passively replicated is observed after stress. This plasticity in origin use is a simple mechanism to recover DNA replication between stalled and collapsed replication forks. The mechanism(s) that limits origin firing to one per replicon in the absence of stress is not known. We recently reported that the DNA damage signaling kinases ATR and Chk1 inhibit activation of the replicative helicase in the absence of stress. In preliminary studies, we show that Chk1 kinase activity is strictly associated with ATR kinase-dependent phosphorylations on Chk1 and that these have an astonishingly short half-life in cells. We propose that this is a highly innovative mechanism that localizes Chk1 kinase activity to the immediate vicinity of ATR at active replicative helicases. We also show that Rif1, which has been implicated in the regulation of replication timing previously, is phosphorylated and that phosphorylated Rif1 binds protein phosphatase 1α (PP1α). Based upon these findings, we hypothesize that Chk1 kinase activity generates a ring of Rif1-PP1α around active replicative helicases and that this limits Cdc7 kinase-dependent origin firing across a replicon. In Aim 1 we will investigate a new mechanistic paradigm for localizing DNA damage signaling to a small volume of the nucleus in the absence of stress. In Aim 2 we will investigate the molecular mechanism that limits activation of the replicative helicase across a replicon in the absence of stress. In Aim 3 we will investigate the impact of ATR kinase inhibition and conformal radiation on immune responses in tumor bearing mice. These studies are highly impactful as they will identify a fundamental mechanism that determines inter-origin distance, genome stability and the rate of cell division in higher eukaryotes. Since this mechanism may be attenuated in T cells, these studies will provide fundamental insights into adaptive immune responses. Our studies may have an immediate impact as the ATR and Chk1 kinase inhibitors used here are in clinical trials.