Cancer Biology

In the human cell cycle, complete replication of DNA is a fundamental process for the maintenance of genome integrity. Replication stress interfering with the progression of replication forks causes difficult-to-replicate regions to remain under-replicated until the onset of mitosis. In early mitosis, a homology-directed repair DNA synthesis, called mitotic DNA synthesis (MiDAS), is triggered to complete DNA replication. Here, we present a method to detect MiDAS in human U2OS 40-2-6 cells, in which repetitive lacO sequences integrated into the human chromosome evoke replication stress and concomitant incomplete replication of the lacO array. Immunostaining of BrdU and LacI proteins is applied for visualization of DNA synthesis in early mitosis and the lacO array, respectively. This protocol has been established to easily detect MiDAS at specific loci using only common immunostaining methods and may be optimized for the investigation of other difficult-to-replicate regions marked with site-specific binding proteins.

During mitosis chromosomes are condensed into dense X-shaped structures that allow for microscopic determination of karyotype as well as inspection of chromosome morphology.

This protocol describes a method to perform immunostaining of formaldehyde-fixed metaphase chromosomes from the avian cell line DT40. It was developed to characterize the localization of YFP-tagged TopBP1 on mitotic chromosomes and specifically determine the percentage of TopBP1 foci that formed on breaks/gaps as well as ends of individual metaphase macrochromosomes (Pedersen et al., 2015). For this purpose immunostaining of YFP was applied. However, the protocol may be optimized for other cell lines or epitopes.

Fluorescence Activated Cell Sorting (FACS) is used to study DNA cell content. Propidium iodide (PI) intercalates into double-stranded nucleic acids and fluoresces. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. Thus PI staining is included in immunofluorescent staining protocols to identify dead cells. DNA staining can be used to study the cell cycle. Relative DNA content shows the proportion of cells in G1, G2 and S phases. Apoptotic cells show characteristic smear on DNA staining. Here a protocol to stain cells by PI is described.