FIG. 4.
Nse5/6 are novel nuclear factors that prevent spontaneous DNA damage and facilitate repair/segregation of DNA. (A) Nse5 and Nse6, myc tagged at their genomic loci, were detected in cells by indirect immunofluorescence with a monoclonal anti-myc antibody. (B) The DNA structure checkpoints are constitutively active in Nse6 mutants, and chromosomes fail to segregate properly after DNA damage. In the top two panels, wild-type (Wt) cells are shown without treatment (left) and after UV irradiation at 100 J/m2 (right). Nuclei stained with DAPI (4′,6′-diamidino-2-phenylindole) have been merged with the corresponding phase images to more clearly show cell cycle stage and mitotic abnormalities. Nse6 mutant cells (nse6Δ) are shown below the wild type. Arrowheads indicate aberrantly long cells (left) or mitotic abnormalities after UV irradiation (right). The last two panels show double mutants of nse6Δ with enforcers of the DNA structure checkpoints, Chk1 and Rad3, in the absence of extrinsic DNA damaging agents. Arrowheads indicate aberrant mitoses, including aneuploid or “cut” cells. (C) Nse6 mutants exhibit high levels of spontaneous DNA damage, as judged by the formation of foci of the HRR factor Rad22. Quantification of the percentage of wild-type or nse6Δ cells containing at least one Rad22-YFP focus, in the absence of extrinsic DNA-damaging agents.