DNA damage in cells occurs from both endogenous and exogenous sources, and failure to repair such damage is associated with the emergence of different cancers, neurological disorders and aging. DNA damage responses (DDR) in cells are closely associated with the cell cycle. While most of our knowledge of DDR comes from bulk biochemistry, such methods require cells to be arrested at specific stages for cell cycle studies, potentially altering measured responses; nor is cell to cell variability in DDR or direct cell-level correlation of two response metrics measured in such methods. To overcome these limitations we developed a microscopy-based assay for determining cell cycle stages over large cell numbers. This method can be used to study cell-cycle-dependent DDR in cultured cells without the need for cell synchronization. Upon DNA damage γH2A.X induction was correlated to nuclear enrichment of p53 on a cell-by-cell basis and in a cell cycle dependent manner. Imaging-based cell cycle staging was combined with single molecule P53 mRNA detection and immunofluorescence for p53 protein in the very same cells to reveal an intriguing repression of P53 transcript numbers due to reduced transcription across different stages of the cell cycle during DNA damage. Our study hints at an unexplored mechanism for p53 regulation and underscores the importance of measuring single cell level responses to DNA damage.

Ultraviolet (UV) radiation is a major environmental mutagen. Exposure to UV leads to a sharp peak of γH2AX – the phosphorylated form of a histone variant H2AX – in the S phase within an asynchronous population of cells. γH2AX is often considered as a definitive marker of DNA damage inside a cell. In this report we show that γH2AX in the S-phase cells after UV irradiation reports neither on the extent of primary DNA damage in the form of cyclobutane pyrimidine dimers nor on the extent of its secondary manifestations in the form of DNA double strand breaks or in the inhibition of global transcription. Instead γH2AX in the S phase corresponds to the sites of active replication at the time of UV irradiation. This accumulation of γH2AX at replication sites slows down the replication. However, the cells do complete the replication of their genomes and arrest within the G2 phase. Our study suggests that it is not DNA damage but the response elicited which peaks in the S phase upon UV irradiation.

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Highlights d CTCF insulates the domains of assorted mono-and biallelically expressed genes d The dosage of mono-allelically expressed genes is more sensitive to CTCF depletion d Active and inactive alleles of mono-allelically expressed genes conform distinctly d CTCF-depletion de-represses inactive alleles of monoallelically expressed genes