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.
| Kaufmann, William K (2010) The human intra-S checkpoint response to UVC-induced DNA damage. Carcinogenesis 31:751-65 |
| Chen, Bo; Simpson, Dennis A; Zhou, Yingchun et al. (2009) Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53. Cell Cycle 8:1775-87 |
| Unsal-Kacmaz, Keziban; Chastain, Paul D; Qu, Ping-Ping et al. (2007) The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement. Mol Cell Biol 27:3131-42 |
| Heffernan, Timothy P; Unsal-Kacmaz, Keziban; Heinloth, Alexandra N et al. (2007) Cdc7-Dbf4 and the human S checkpoint response to UVC. J Biol Chem 282:9458-68 |
| Chastain 2nd, Paul D; Heffernan, Timothy P; Nevis, Kathleen R et al. (2006) Checkpoint regulation of replication dynamics in UV-irradiated human cells. Cell Cycle 5:2160-7 |
| Kaufmann, William K; Heffernan, Timothy P; Beaulieu, Lea M et al. (2003) Caffeine and human DNA metabolism: the magic and the mystery. Mutat Res 532:85-102 |
