CYCLIN D1-MEDIATED INHIBITION OF REPAIR AND REPLICATIVE DNA-SYNTHESIS IN HUMAN FIBROBLASTS

Cyclin D1 is a key regulator of the G(1) phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G(1). Cyclin D1 is localized to the nucleus during G(1). We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G(1) cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication, Cyclin D1 appears to be an essential component of a G(1)-checkpoint.