TopBP1-mediated DNA processing during mitosis

Abstract

Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

Keywords: PCNA ubiquitylation; REV1; TopBP1; fork restart; mitosis; polymerase η; post-replicative gaps; replication stress; translesion synthesis.

Figure 1.

TopBP1 mediates unscheduled DNA synthesis in mitosis independently of TLS pathways. (A) Representative images of fixed DT40 cells (TopBP1^mCherry) treated with 0.4 μM APH for 20 h and pulse labeled with 20 μM EdU for 20 min. White arrowheads indicate TopBP1 focus colocalizing with EdU. (B) Quantification of EdU in prometaphase and metaphase cells. (C) Representative images of an APH-treated DT40 cell line (TopBP1^{YFP-AID/YFP-AID/YFP-AID}/ osTIR / TFP-hPol η / hH2B-mCherry). (D) Quantification of TopBP1 and pol η foci. Live cell images were captured of untreated cells or 20 h after addition of 0.4 μM APH. TopBP1 and pol η foci and their colocalization in mitotic cells were quantified. (E) Representative images of an APH-treated DT40 cell line (FANCD2^Venus/Venus/ TFP-hPol η). (F) Quantification of FANCD2 and pol η foci. Live cell images were captured of untreated cells or 20 h after addition of 0.4 μM APH. FANCD2 and pol η foci and their colocalization in mitotic cells were quantified. For all graphs in this figure, asterisks indicate significant differences from the untreated (P < 0.05) and error bars represent 95% confidence intervals. Number of cells analyzed is indicated (n).