S Checkpoint Function in Human Fibroblasts

描述（由申请人提供）：该项目涉及一种称为S检查点的细胞监视途径，当DNA受损时，S检查点会抑制DNA合成。速度沉降分析表明，这种抑制是由复制子起始速率的降低引起的。S期人类细胞中复制子的起始过程似乎受到细胞周期蛋白依赖性激酶2（Cdk 2）、Dbf 4依赖性激酶（Ddk）和Cdc 6的正向调节。来自患有共济失调毛细血管扩张症（AT）的患者的细胞由于ATM中的失活突变而在电离辐射（IR）诱导的S检查点功能中有缺陷。Nijmegen断裂综合征（NBS）患者由于NBS 1的突变而具有类似的S检查点功能缺陷。与这些观察结果雅阁，已经显示ATM激酶响应于DNA损伤而磷酸化NBS 1。我们推测，DNA损伤IR诱导ATM磷酸化NBS 1和其他效应底物，以抑制Cdk 2和Ddk在S期细胞，从而抑制DNA合成的起始在复制起点。为了检验这一假设，我们将量化IR诱导的DNA合成抑制在确定的复制子起源在二倍体人成纤维细胞永生化的端粒酶表达。将对AT、NBS和AT样细胞进行类似测试，以确定辐射诱导的复制子起始抑制是否依赖于ATM、NBS 1和MRE-1基因产物。Cdk 2和Cdc 6协同启动Gi核中DNA复制的无细胞系统将用于体外测定S检查点功能。我们将测试Cdk 2和Cdc 6是否需要启动DNA复制在真正的复制子在分离的Gi细胞核的起点。然后将检查该体外起始系统以确定用IR处理细胞核是否激活ATM依赖性信号传导途径，从而导致Cdk 2和复制子起始的抑制。完整细胞的研究将确定Cdk 2和Ddk是否在辐照后被抑制，其动力学相当于复制子起始的抑制。改变的Cdc 25 C和Cdk 2等位基因也将在二倍体人成纤维细胞中表达，以评估Cdk 2的活性位点磷酸化在S检查点应答中的作用。该项目将定义S检查点的遗传组成部分，并阐明二倍体人成纤维细胞中S检查点反应的信号转导机制。

DESCRIPTION (provided by applicant): This project concerns a cellular surveillance pathway known as the S checkpoint that inhibits DNA synthesis when DNA is damaged. Velocity sedimentation analyses indicate that this inhibition is brought about by reduction in the rate of replicon initiation. The process of initiation of replicons in S phase human cells appears to be positively regulated by cyclin-dependent kinase 2 (Cdk2), Dbf4-dependent kinase (Ddk) and Cdc6. Cells from patients with ataxia telangiectasia (AT) are defective in ionizing radiation (lR)-induced S checkpoint function due to inactivating mutations in ATM. Patients with Nijmegen Breakage Syndrome (NBS) have a similar defect in S checkpoint function due to mutations in NBS1 In accord with these observations, it has been shown that ATM kinase phosphorylates NBS1 in response to DNA damage. We postulate that DNA damage by IR induces ATM to phosphorylate NBS1 and other effector substrates to inhibit Cdk2 and Ddk in S phase cells, thereby inhibiting initiation of DNA synthesis at origins of replication. To test this hypothesis we will quantify the lR-induced inhibition of DNA synthesis within defined replicon origins in diploid human fibroblasts immortalized by expression of telomerase. AT, NBS, and AT-like cells will be similarly tested to determine whether radiation-induced inhibition of replicon initiation is dependent upon the ATM, NBS1 and MRE-1 gene products. A cell-free system in which Cdk2 and Cdc6 cooperate to initiate DNA replication in Gi nuclei will be used to assay for S checkpoint function in vitro. We will test whether Cdk2 and Cdc6 are required to initiate DNA replication at bona fide replicon origins in isolated Gi nuclei. This system of in vitro initiation will then be examined to determine whether treatment of nuclei with IR activates ATM-dependent signaling pathways leading to inhibition of Cdk2 and replicon initiation. Studies with intact cells will determine whether Cdk2 and Ddk are inhibited post-irradiation with kinetics equivalent to the inhibition of replicon initiation. Altered Cdc25C and Cdk2 alleles also will be expressed in diploid human fibroblasts to assess the role of active-site phosphorylation of Cdk2 in S checkpoint response. This project will define genetic components of the S checkpoint and elucidate signal transduction mechanisms that underlie S checkpoint response in diploid human fibroblasts.