BBB seminar: Stig-Ove Bøe
A role of PML-nuclear bodies in DNA replication and the cellular response to DNA damage
Section of Anatomy and Cell Biology, Department of Biomedicine, University of Bergen
The mammalian nucleus is organized into several distinct compartments that support different cellular possesses such as DNA replication, gene expression and maintenance of genome integrity. Our laboratory is interested in elucidating the function of a particular nuclear compartment termed the PML-nuclear body (PML-NB). This nuclear structure has been named after the promyelocytic leukaemia (PML) protein, a tumour suppressor protein, which is the only component so far that has been shown to be absolutely required for the assembly of PML-NBs. A normal cell commonly contains between 20 and 30 of these structures, and they have been shown to play important roles in cellular functions such as apoptosis, transcription regulation, differentiation and maintenance of genome stability.
Using an experimental approach based on SV40 large T-antigen mediated plasmid replication we have demonstrated that PML-NBs have the capacity to sequester newly synthesized DNA molecules. Recruitment of DNA into this compartment depended on active plasmid DNA synthesis, and a significant portion of the accumulated DNA was found to be single-stranded. Using a single-strand DNA (ssDNA) detection assay we have also demonstrated that PML-NBs recruit single-stranded chromosomal DNA in response to chromosomal DNA damage. The accumulation of ssDNA in this case was strongly enhanced by caffeine, a drug that is known to promote excessive firing of replication origins and to inhibit the activation of cell cycle checkpoints. Small interference RNA (siRNA)-mediated depletion of PML led to a prominent S-phase arrest following treatment of cells with DNA damaging agents. Furthermore, siRNA mediated suppression of proteins with known functions in replication fork stabilization significantly enhanced the PML dependent accumulation of ssDNA in PML-NBs. Our observations demonstrate a functional link between PML-NBs, the process of DNA replication and the cellular response to DNA damage. A model will be discussed where PML-NBs participate in the repair of collapsed replication forks and re-initiation of DNA syntheses in situations where the DNA replication machinery encounters damaged DNA.