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In response to acute DNA damage, proliferating cells can change their cell cycle progression through the DNA damage checkpoint in the DNA damage response (DDR) pathway. The DDR pathway is largely conserved but differs in different cell cycle

phases. Therefore, it is possible that the cell fate decision in response to DNA damage can vary depending on the cell cycle phase during which the damage occur 8,94_{. In} particular, while acute DNA damage can halt the cell cycle phase progression

throughout most of the cell cycle, DNA damage during replication does not arrest the cell cycle in S phase8_{. Instead, cells that experience damage postpone arrest until} finishing DNA replication. The implication of this delayed cell cycle respond on the cell fate is not completely understood.

A central responder of the DDR is p21, a potent inhibitor of cyclin-dependent kinase (CDK). DNA damage-activated p21 induction leads to impediment of the cell cycle progression. However, during S phase, p21 is subject to PCNA-dependent CUL4- Cdt2 degradation38,39 _{(Figure 4.1A), making S phase progression the least sensitive to} acute DNA damage8_{. When DNA replication is completed, p21 is able to accumulate} and exerts its effect on CDK. How S-phase exit affects the systems-level dynamics of cell cycle regulators that eventually lead to differential cell fate decisions is unknown.

To investigate the cell fate of proliferating cells that experience acute DNA damage during S phase, we induced DNA double strand breaks (DSBs) using

neocarzinostatin (NCS) and followed the cell cycle distribution (Figure 4.1B). To distinguish cells damaged in S phase, we used a EdU pulse to label S cells. We found that the majority of the S-phase damaged cells were arrested in the subsequent G2 in the 2N chromosome state and remained 2N for at least 48 hours (Figure 4.1B, Figure S4.1A). It has been known that DNA damage can cause mitosis skip, leading to a tetraploid G1-like senescent state134,149_{. Because the G2 arrested population remained} stable, it is likely that these cells have entered the irreversible senescent state. Indeed, SA-β-gal staining, a gold standard assay for senescence, revealed that most S-phase damaged cells entered senescence (Figure 4.1C-D). Although the majority of cells entered senescence in response to this NCS dosage, a small proportion of the damaged cells had completed mitosis and remained non-senescent.

4.4.2 The mitosis-senescence decision is controlled by a bifurcation in CDK activity at S-phase exit

We next asked what determined the cell fate decision—whether to enter

senescence or mitosis—upon acute DSB damage in S phase. The progression through cell cycle is largely promoted by the cyclin-CDK activity, and this activity has been shown to determine the proliferation-quiescence decision and passage through the restriction point34,150_{. To determine whether the mitosis-senescence decision is}

associated with differential CDK activity levels, we introduced a CDK activity reporter34 (DHB-mCherry) and a S-phase reporter (PCNA-mTurqoise)8,84 _{and monitored CDK} activities in single cells upon DNA damage and S-phase exit (Figure 4.2A, Figure S4.1B-C). We found that when DSBs were introduced during S phase, CDK activity level bifurcated into a high-CDK and a low-CDK activity state, corresponding to cell fate of mitosis and senescence, respectively (Figure 4.2B). Surprisingly, the bifurcation in

CDK activity mainly occurred before the S/G2 transition (Figure 4.2B-C), suggesting that the decision to enter mitosis or senescence was made during S phase. Because p21 was unable to accumulate to suppress CDK activity during S phase, the CDK activity could not reveal the differential responses to the DNA damage until exiting S- phase. However, the immediate bifurcation at S-phase exit suggested that cells already decoded the DNA damage response into a cell-fate decision during S phase, and this decision was not disclosed until p21 was released from replication-associated

degradation to suppress CDK activity38,39_{. We also observed similar bifurcation behavior} at S-phase exit under replication stress (Figure 4.2D). However, unlike the NCS-

induced DSB damage, the aphidicolin-induced replication stress damage was able to immediately suppress CDK activity via the ATR-Chk1-Wee1 pathway, even in the absence of p21 (Figure 4.2D)30,35_.

4.4.3 The mitosis-senescence decision is contributed by the DNA damage repair, but not the timing of damage or the damage level incurred.

We next asked what causes the cell-to-cell heterogeneity in cell fate in response to DNA damage during S phase. Previous studies have shown that the timing of DNA damage within a cell cycle can lead to different cell-fate outcomes8,94_{. To test whether} the timing within S phase that the DSBs were incurred affected the decision to mitosis or senescence, we measured how far into S phase did the damaged occurred in the mitosis and senescence groups (Figure 4.3A). We did not detect a significant difference in damage timing between the two groups (Figure 4.3B, left panel), suggesting the timing when the damage occurred in S phase did not contribute to cell fate. However, the senescent group showed a prolonged S phase after damage compared to the

mitotic group (Figure 4.3B, right panel), indicating that there was already a differential response to DNA damage before S-phase exit.

DNA damage has been suggested to be one of the major factors determining the proliferation-quiescence decision7,22,31,93_{. Therefore, we then investigated whether the} level of DNA damage correlated with the cell fates. To quantify DNA damage, we introduced into our cell line a fluorescence-tagged 53BP1, a DDR protein that localizes to sites of DSBs, as a reporter for DNA double strand breaks (DSBs)151 _{(Figure 4.3C,} Figure S4.2A-D). Upon NCS treatment, the number of 53BP1 foci immediately

increased (Figure 4.3C). When we compared the 53BP1 foci number in the mitosis and senescence populations, we observed on average a lower amount of 53BP1 foci in the mitosis population after damage (Figure 4.3D-E). This difference in DSB level could be contributed by the different amount of DSBs induced, or by the different DSB repair rate. We found no difference between the amount of 53BP1 foci number induced upon NCS treatment (Figure 4.3F), indicating that the mitosis-senescence decision was

contributed by the different levels of initial DSBs. In contrast, the repair rate of 53BP1 foci was significantly faster in the mitosis population than in the senescence population (Figure 4.3G, Figure S4.2E), suggesting that slower damage repair rate was

associated with higher probability of entering senescence.

To determine if a slower damage repair rate indeed contributed to senescence, we decreased the DSB repair rate by treating cells with a small molecule inhibitor (NU- 7441) highly specific for DNA-PK152_{, an enzyme required for non-homologous end} joining (NHEJ) repair pathway. Co-treating cell with DNA-PK inhibitor in addition to NCS prolonged the 53BP1 foci half-lives in the population (Figure 4.3H) as well as in single

cells (Figure 4.3I). In addition, addition of DNA-PK inhibitor in S phase-damaged cells steered the cell fate towards senescence and completely abolished the mitosis

population (Figure 4.3J), suggesting the rate of DSB repair could determine the cell fate decision.

4.4.4 The CDK activity bifurcation is mediated by p21 level bifurcation and is