S Checkpoint Function in Human Fibroblasts

描述(申请人提供)：这个项目涉及一种被称为S检查点的细胞监测途径，当dna受损时，它会抑制dna合成。速度沉降分析表明，这种抑制是由于复制子启动速度的降低所带来的。细胞周期蛋白依赖性蛋白激酶2(CDK2)、Dbf4依赖性蛋白激酶(DDK)和CDC6正向调控着S期人细胞中复制子的启动过程。共济失调毛细血管扩张症(AT)患者的细胞在电离辐射(LR)诱导的S检查点功能上存在缺陷，这是由于ATM中的突变失活所致。奈梅根断裂综合征患者的S检查点功能也有类似的缺陷，原因是Nbs1基因突变。与这些观察结果一致，已有研究表明，ATM激酶对Nbs1的磷酸化是对DNA损伤的反应。我们推测，IR引起的DNA损伤诱导ATM磷酸化NBS1和其他效应底物，从而抑制S期细胞中CDK2和DDK的表达，从而抑制复制起始时DNA合成的启动。为了验证这一假设，我们将量化LR对通过表达端粒酶而永生化的二倍体人成纤维细胞中特定复制子来源的DNA合成的抑制。AT、NBS和类似AT的细胞将进行类似的测试，以确定辐射诱导的复制子启动抑制是否依赖于ATM、NBS1和MRE-1基因产物。利用CDK2和CDC6协同作用在胃肠道细胞核内启动DNA复制的无细胞系统，将用于体外检测S检查点功能。我们将测试在分离的胃肠道细胞核中是否需要CDK2和CDC6在真正的复制子起始处启动DNA复制。这一体外启动系统将被用来确定IR处理细胞核是否激活了ATM依赖的信号通路，从而抑制了CDK2和复制子的启动。对完整细胞的研究将确定CDK2和DDK在辐射后是否受到抑制，动力学相当于抑制复制子的启动。改变的CDC25C和CDK2等位基因也将在二倍体人成纤维细胞中表达，以评估CDK2的活性部位磷酸化在S检查点反应中的作用。该项目将定义S检查点的遗传成分，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.