PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

Abstract

PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localized enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilized an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol(-/-)) on the downstream maintenance of cells after UV damage. We report that PrimPol(-/-) cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol(-/-) arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol(-/-) cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase.

Keywords: Chk1; DT40; PrimPol; TLS; UV; cell cycle; checkpoint; polymerase; primase; replication.

Figure 1.

PrimPol^−/− cells show decreased UV-C sensitivity with dose compared to wild type and Pol η^−/− in viability but not clonal survival assays. (A) Cell viability was measured after increasing doses of UV-C (48 hrs after damage) using Cell Titer Blue, lines represent an average of 3 repeats. (B) Cells were grown in the presence of increasing doses of 4NQO or media alone for 48 hrs followed by viability analysis with Cell Titer Blue, or after 48 hrs they were washed with PBS and grown for a further 72 hrs in media alone before viability analysis (C). This method was compared with clonal cell survival after UV-C, where cells were plated singularly after increasing doses of UV-C irradiation and growth to form a colony was counted (n = 3) (D). Significance was calculated using a students T-test, at 2 J/m² p < 0.05 WT: PrimPol^−/− cl1, WT: Pol η^−/− and PrimPol^−/− cl2 p < 0.01. Error bars represent standard deviation of repeat experiments in all cases.

Figure 2.

UV-C damage causes extended G2 arrest in PrimPol^−/− cells leading to decreased cell death but increased aberrant mitotic division. (A) Cells were stained with DAPI and normal nuclei populations were compared for the percentage of fragmented nuclei 16 hrs after UV-C damage, n ≥ 3 independent experiments and error bars represent standard deviation. (B) Cells were also co-stained with α-tubulin to identify mitotic cells with multipolar spindles, example images (16 hrs after 2 J/m² UV-C) (scale bar 10 µM). Quantification (16 hrs after 4 J/m² UV-C) is shown in (C). (D) Cells were analyzed by FACS after propidium iodide staining at increasing recovery time-points after 4 J/m² UV-C damage, average G2/M population is shown from 3 independent experiments. (E) Mitotic entry was analyzed by p-H3 staining during a 4 hr nocodozole treatment, 0 or 16 hrs after 0 or 4 J/m² UV-C damage. (F) Cells unable to undergo replication during a 16 hr EdU labeling were counted after 0 or 4 J/m² UV-C followed by a 24 hr recovery period, representative images shown in Figure S2C. In all cases error bars represent standard deviation and significance was measured using an unpaired students T-test (* p < 0.05, ** p < 0.01, ***p,0.001).

Figure 3.

UV-C induced checkpoint activation in PrimPol^−/− cells is partially resolved by inhibition of Chk1 or p38. (A) Chk1 phosphorylation was analyzed by western blotting of whole cell lysates at increasing recovery times (2-24 hrs) after 4 J/m² UV-C damage. (B) The affect of UCN-01 on cell cycle progression was measured by counting the presence of p-H3 positive mitotic cells. Cells were pre-treated with 100 nM UCN-01 for approximately 2 hrs before irradiation with 0 or 4 J/m² UV-C, cells were allowed to recover for 0 or 16 hr before the addition of nocadozole to block mitotic exit for 4 hrs. (C) Mitotic segregation was analyzed by staining with DAPI and α-tubulin 16 hrs after cells were damaged with 4 J/m² in this case cells were pre-treated and then maintained in 100 nM UCN-01 prior to damage. (D) Effect of p38 on cell cycle progression was measured by counting the percentage of p-H3 positive mitotic cells 4 hrs after incubation with nocodazole. Cells were first pre-treated with 2.5 μM SB203580 for 2 hrs followed by irradiation with 4 J/m² UV-C, and a 16 hr recovery period. (E) The ability of checkpoint inhibitors to release cells from G2 arrest was measured by allowing cells to recover after 0 or 4 J/m² UV-C for 16 hrs, followed by addition of 100 nM UCN-01 or 2.5 µM SB203580 and 0.5 µM nocodazole for 4 hrs. Mitotic entry was then assessed by p-H3 staining. For all experiments n ≥ 3 independent experiments, error bars represent standard deviation.

Figure 4.

PrimPol^−/− cells are more resistant to Chk1 inhibition than WT cells. (A) Cell viability was measured using Cell Titer Blue, 48 hrs after 4 J/m² UV-C damage, where cells were maintained in 100 nM UCN-01. (B) Colony formation was analyzed in the presence of 2 mM caffeine after increasing doses of UV-C damage in comparison with survival in the absence of the inhibitor. (C) Cell Titer Blue was used to measure cell viability 48 hrs after 4 J/m² UV-C damage, where cells were pre-treated and maintained in 2.5 µM of SB203580, p38 inhibitor. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).

Figure 5.

Decreased rates of cell cycle progression are protective in PrimPol^−/− cells. (A) Cell death over time was followed by bright field live cell imaging of H2B RFP labeled cells (WT and PrimPol^−/−), after 4 J/m² UV-C damage with or without the presence of 100 nM UCN-01 by counting dead cells as a percentage of the whole population. (B) DT40 cells were followed by live cell imaging after UV-C, allowing the point of cell death to be observed. Cell death was quantified dependent on whether the cell had undergone mitosis prior to death. (C) The effect of the UCN-01 inhibitor on cell cycle populations was analyzed by flow cytometry on propidium iodide stained cells after 24 hrs incubation with 100 nM UCN-01 in the absence of damage. (D) Cell cycle progression rates were measured by analysis of the number of S-phase cells marked by an EdU pulse that were able to progress into mitosis, identified by p-H3 staining in a 4 hr period after 0 or 4 J/m² UV-C damage. Error bars represent standard deviation of independent repeats and significance was analysed using a students T-test (*p < 0.05, **p < 0.01).

Figure 6.

Cell cycle progression in verterbrate cells after UV-C damage. A schematic model showing the possible outcomes as a cell progresses through the cell cycle after UV-C damage, in comparison with undamaged cells. Work described here has identified differences in the percentage of cells achieving each outcome, dependent on its complement of TLS polymerases. An increase in Outcome 4 is observed in the absence of PrimPol, while Outcome 3 becomes more prevalent in cells lacking Pol η.