S Checkpoint Function in Human Fibroblasts

描述（由申请人提供）：该项目涉及被称为S检查点的细胞监视途径，当DNA受损时抑制DNA合成。速度沉降分析表明，这种抑制作用是由复制子起始速率的降低引起的。S期人细胞复制子的启动过程似乎受到周期蛋白依赖性激酶2 （Cdk2）、dbf4依赖性激酶（Ddk）和Cdc6的正调控。来自共济失调毛细血管扩张（AT）患者的细胞在电离辐射（lR）诱导的S检查点功能中由于ATM失活突变而存在缺陷。奈梅根断裂综合征（NBS）患者由于NBS1的突变在S检查点功能上也有类似的缺陷。与这些观察结果一致，已经证明ATM激酶在DNA损伤时磷酸化NBS1。我们假设IR的DNA损伤诱导ATM磷酸化NBS1和其他效应底物，从而抑制S期细胞中的Cdk2和Ddk，从而抑制复制起始处DNA合成的起始。为了验证这一假设，我们将在端粒酶表达永生的二倍体人成纤维细胞中量化lr诱导的DNA合成抑制。将对AT、NBS和AT样细胞进行类似的测试，以确定辐射诱导的复制子起始抑制是否依赖于ATM、NBS1和MRE-1基因产物。Cdk2和Cdc6协同启动Gi细胞核DNA复制的无细胞系统将用于体外S检查点功能的测定。我们将测试在分离的Gi细胞核中，Cdk2和Cdc6是否需要在真正的复制子起源处启动DNA复制。然后将检查这种体外起始系统，以确定用IR处理细胞核是否激活atm依赖的信号通路，从而抑制Cdk2和复制子起始。对完整细胞的研究将确定Cdk2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活性位点磷酸化在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，并阐明二倍体人类成纤维细胞中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.