The Role of Nuclear Architecture in DNA Repair

Abstract

Precise replication of genetic material and the maintenance of genome integrity by DNA repair mechanisms are critical processes in all organisms. The inaccurate repair or accumulation of unrepaired errors can overwhelm the cell resulting in senescence, apoptosis or cancer development. DNA lesions must be identified and efficiently repaired in the context of a highly compartmentalized nucleus, consisting of discrete chromosome territories interspersed among many specialized, functionally distinct subnuclear domains. Disruption of some of these domains or of nuclear structural components is known to result in increased genomic instability. To further understand the contributions of nuclear architecture and organization to the maintenance of genomic integrity, I investigated the roles of polymerized nuclear actin, the nuclear lamina and PML nuclear bodies on DNA repair efficiency. I identified a novel role for nuclear actin in the non-homologous end-joining pathway of DNA repair. Altering the polymerization state of nuclear actin was found to negatively impact the retention of the GFP-tagged damage-sensing protein Ku80 at sites of DNA damage in live cells. Additionally, I examined DNA repair efficiency at the nuclear lamina in relation to chromatin ultrastructure using electron microscopy, finding that the delay in DNA repair at this nuclear compartment was due to associated heterochromatin. Finally, I investigated the role of PML isoforms and PML nuclear bodies in DNA repair. Using genome engineering, I created versatile cell lines containing a single copy of a DNA repair reporter integrated at defined nuclear locations. Using these cell lines, homologous recombination was found to be decreased when a DNA break occurred within chromatin significantly associated with PML nuclear bodies compared to an unassociated DNA break. Additionally, using these cell lines along with wild-type U2OS and a U2OS PML knockout cell line, I demonstrated that PML isoform overexpression, notably that of PML I, II and IV, leads to significant inhibition of both homologous recombination and non-homologous end joining, suggesting an early role for PML/PML nuclear bodies in DNA repair. Collectively, these results demonstrate a complex involvement of nuclear organization and DNA repair, and that the maintenance of genomic stability is intimately linked to nuclear architecture.