Figure 7 .

Spd1 protects cells from division and mutation during dNTP imbalance. (A) Comparison between wild-type and spd1Δ hsv-tk⁺ hENT⁺ strains by spot test on EMM plates containing BrdU, EdU, or thymidine. DMSO is a vehicle control for EdU. Shown is the minimal dose where wild-type cells began to show sensitivity to analogs. Strains FY2317, -3454, and -6247 are shown. (B) Cultures were treated with 32.6 µM BrdU and plated to calculate viability relative to 0 hr. Wild-type (wt) and spd1Δ strains express hsv-tk⁺ hENT⁺ (FY2317 and -6247), while the nonincorporating control (non-inc, FY3454) does not. Shown are mean viability values from three independent experiments ± SEM. (C) Proliferation was monitored by counting cell concentration during BrdU treatment for cultures as in A, in addition to rad3Δ hsv-tk⁺ hENT⁺ (FY5150). Shown are mean viability values (n = 3 experiments) ± SEM. (D) Canavanine mutation was scored for incorporating wild-type and spd1Δ strains (FY2317 and -6247), with or without 32.6 µM BrdU treatment (2 hr, 32°). Lea and Coulson fluctuation analysis was used to calculate the rate of can1⁻ forward mutation (per 10⁷ generations) in independent cultures over three experiments (wt, n = 12; spd1Δ, n = 15). Shown are median mutation rates with quartile bounding boxes and 95% C.I. error whiskers. Significance was assessed by two-tailed pairwise Mann–Whitney U-tests, *P = 0.0001, **P < 0.0001. (E) Colonies of wild-type or spd1Δ cells, untreated (no drug) or following 2 hr in 32.6 µM BrdU, were grown on YES and then replicated onto medium with FUdR to score for hsv-tk⁺ loss. Significance was assessed by two-tailed Z-test (**P < 0.0002). (F) Enhanced sectoring of colonies on FUdR was noted for spd1Δ cells either untreated or following BrdU exposure as in E (two-tailed Z-test, ** P < 0.0002), compared to BrdU-treated wild-type cells. Inset, example of spd1Δ colony with hsv-tk⁺ loss (*) or sectored area (arrow). Frequencies were calculated from independent experiments, presented with 95% C.I. (G) Model for the effect of exogenous thymidine (Thy) and nucleoside analogs in fission yeast cells expressing a reconstituted thymidine salvage pathway (hsv-tk⁺). Details are in Discussion.