DNA damage signaling to dormant origins of replication

DNA 损伤向休眠复制起点发出信号

• 批准号: 10295771
• 负责人: CHRISTOPHER J. BAKKENIST
• 金额: $ 35.08万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10295771
• 关键词: Attenuated; Binding; Binding Proteins; CD8-Positive T-Lymphocytes; CDC2 gene; CDC7 gene; CHEK1 gene; Cancer Model; Cell Lineage; Cell Nucleus; Cell division; Cells; Chromosomal Instability; Chromosome 1; Chromosomes; Clinic; Clinical Trials; Complement; Conformal Radiotherapy; DNA Damage; DNA biosynthesis; DNA replication fork; DNA replication origin; Data; Diffuse; Eukaryota; Fire - disasters; Fluorescence; Fluorescence Resonance Energy Transfer; Genome; Genome Stability; Half-Life; Human; Human Chromosomes; Human Genome; Immune response; Immunotherapy; In Vitro; Irradiated tumor; Length; Medical Oncology; Microscopy; Molecular; Molecular Conformation; Mus; Normal Cell; Pattern; Phosphorylation; Phosphotransferases; Proliferating; Protein phosphatase; Radiation; Radiation Oncology; Regulation; Replication Origin; Replicon; Reporter; Reporting; Resolution; Role; S phase; Signal Transduction; Stress; T-Lymphocyte; Testing; Time; adaptive immune response; base; cancer cell; helicase; in vivo; innovation; insight; kinase inhibitor; lymph nodes; mouse model; novel; radiation response; treatment response; tumor

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.

高等真核生物进化的机制，启动DNA复制在多个起源， 染色体在约50，000个复制子中的每一个中，在单个起点处激活复制解旋酶是足够的 在没有压力的情况下复制人类基因组这约50，000个起源是从五到五个来源中选出的。 超过许可来源的十倍。激活额外的复制解旋酶的起源，否则 被动复制后观察应力。这种起源使用的可塑性是一种简单的恢复机制 停滞和崩溃的复制叉之间的DNA复制。将原点触发限制为一个的机制 不存在应激时每个复制子的存活率是未知的。我们最近报道了DNA损伤信号激酶 ATR和Chk 1抑制在没有应激的情况下复制解旋酶的活化。在初步研究中，我们 显示Chk 1激酶活性与Chk 1上ATR激酶依赖性磷酸化密切相关， 它们在细胞中的半衰期非常短。我们认为这是一个高度创新的机制 其将Chk 1激酶活性定位于ATR附近的活性复制解旋酶。我们还表明 Rif 1，这已经牵连在调节复制时间之前，是磷酸化的， 磷酸化的Rif 1结合蛋白磷酸酶1α（PP 1 α）。基于这些发现，我们假设， Chk 1激酶活性在活性复制解旋酶周围产生Rif 1-PP 1 α环，这限制了Cdc 7 激酶依赖性的起始点在复制子上的发射。在目标1中，我们将研究一种新的机械范式， 在没有压力的情况下，将DNA损伤信号定位到细胞核的小体积。在目标2中， 研究限制复制解旋酶在复制子中激活的分子机制， 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 荷瘤小鼠的免疫应答。这些研究非常有影响力，因为它们将确定一个基本的 决定起源间距离，基因组稳定性和细胞分裂速率的机制，在较高的 真核生物由于这种机制可能在T细胞中减弱，这些研究将提供基本的见解 适应性免疫反应。我们的研究可能会立即产生影响，因为ATR和Chk 1激酶 这里使用的抑制剂正在临床试验中。