4.4 The DNA damage checkpoint regulates Dpb11-Slx4-dependent Mus81-Mms4
4.4.1 Reduced DNA damage checkpoint activation promotes DNA repair in the
sufficient to restore the function of the Slx4-Dpb11-Mms4-Mus81 complex.
Moreover, premature activation of Mus81-Mms4 might become harmful to cell survival when replication fork stalling occurs in combination with impaired Dpb11-Slx4 interaction in the slx4-S486A mutant.
Figure 4.19. Premature activation of Mms4 does not restore the function of the Dpb11-Slx4 complex.
MMS sensitivity of WT, the slx4-S486A, SS56,184ED and slx4-S486A mms4-SS56,184ED mutants. Cells were spotted in serial dilutions on plates containing MMS. The growth was evaluated after incubation for 2 days at 30°C.
4.4 The DNA damage checkpoint regulates Dpb11-Slx4-dependent
dependent pathway of DNA damage checkpoint activation. Second, we analyzed the ddc1-T602A mutant that is not able to interact with Dpb11 and therefore shows an impaired Dpb11-dependent DNA damage checkpoint activation (Pfander and Diffley, 2011). Third, we took into consideration that tagging of Rad53 with a 3HA epitope also leads to a partially inactive DNA damage checkpoint (Conde et al., 2010). All three DNA damage checkpoint mutants in combination with slx4-S486A were tested for growth on plates containing MMS. Strikingly, all three mutants were able to rescue the slx4-S486A mutant sensitivity to MMS (Figure 4.20a).
Figure 4.20. DNA damage checkpoint regulates the Dpb11-Slx4 complex.
a) MMS sensitivity of WT, the slx4-S486A and dot1Δ deletion, ddc1-T602A, rad53-3HA, rad9Δ deletion mutants and in combination with slx4-S486A. Cells were spotted in serial dilutions on plates containing MMS. The growth was evaluated after incubation for 2 days at 30°C; b) MMS sensitivity of WT, the slx4-S486A, rad9Δ deletion and slx4-S486A rad9Δ mutants. Cells were spotted as in a.
To better define the role of the DNA damage checkpoint for the regulation of the Slx4-Dpb11-Mms4-Mus81 complex function, we tested the effect of complete DNA damage checkpoint inactivation in the slx4-S486A mutant. For this purpose, a strain lacking the DNA damage checkpoint mediator protein Rad9 was constructed.
The rad9Δ deletion mutant is not able to activate the DNA damage checkpoint (Pfander and Diffley, 2011; Ohouo et al., 2013). In contrast to the mutants characterized by a partial inactivation of the DNA damage checkpoint, the rad9Δ deletion mutant was more sensitive to MMS compared to WT cells and the slx4-S486A mutant. Consequently, we did not detect the rescue of the slx4-S486A mutant sensitivity to MMS by deleting RAD9 (Figure 4.20b). These data indicate that a
WT slx4-S486A
dot1Δ slx4-S486A dot1Δ ddc1-T602A slx4-S486A ddc1-T602A rad53-3HA
- MMS 0.025% MMS
slx4-S486A rad53-3HA
0.03% MMS
WT rad9Δ slx4-S486A rad9Δ slx4-S486A
- MMS 0.025% MMS 0.03% MMS
a)
b)
partial but not a complete inactivation of DNA damage checkpoint is beneficial when the Dpb11-Slx4 interaction is impaired.
Figure 4.21. Reduced DNA damage checkpoint activation promotes faster DNA repair progression in the slx4-S486A mutant.
a) Recovery experiment of WT, the slx4-S486A and slx4-S486A ddc1-T602A mutants. Cells were synchronized in G1, released to S-phase in the medium with MMS. After 30 min cells were released to drug-free medium for 3 hours. The samples were taken at different time points and yeast chromosomes were visualized by PFGE. Quantification of the chromosome signal was performed using ImageJ software. The signal intensity in a lane of the gel was normalized to the whole signal including that in a well; b) Recovery experiment as in a. DNA content was measured by FACS; c) Recovery experiment as in a. DNA damage checkpoint activation was evaluated by Western blot using antibodies against Rad53.
WT
Log G1 +MMS 1h 2h 3h
Recovery
FACS
slx4-S486A slx4-S486A ddc1-T602A
WT
Rad53 G1 +MMS 1h 3h2h G1 +MMS 1h 3h2h G1 +MMS 1h 3h2h
anti-Rad53
Recovery Recovery Recovery
slx4-S486A slx4-S486A ddc1-T602A slx4-S486A slx4-S486A ddc1-T602A
G1 +MMS 3h1h 3h
2h+MMS Recovery
1h 3h Recovery
G1 +MMS 2h G1 2h1h
Recovery WT
PFGE
Signal intensity in gel/total
slx4-S486A slx4-S486A ddc1-T602A
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A possible explanation for the rescue of the slx4-S486A mutant sensitivity to MMS by partial DNA damage checkpoint inactivation may be that DNA repair is more efficient under these conditions. To test this hypothesis, we damaged yeast cells with a pulse of MMS and analyzed the chromosomal repair kinetics by pulsed-field gel electrophoresis. Strikingly, we observed that the appearance of chromosomes in the gel was basically identical in the samples of wild type cells and the slx4-S486A ddc1-T602A double mutant. After one hour, WT cells and the slx4-S486A ddc1-T602A double mutant showed the recovery (Figure 4.21a, lanes 3 and 13). In contrast, the slx4-S486A mutant recovered slower, which is in line with data shown previously in figure 4.8a (Figure 4.21a, lanes 6-10). Consistent with the observation obtained by PFGE analysis, DNA replication in the slx4-S486A ddc1-T602A mutant had the same kinetics as in WT in contrast to the slx4-S486A mutant (Figure 4.21b). Finally, we found that the DNA damage checkpoint during the recovery was deactivated faster in the slx4-S486A ddc1-T602A mutant compared to the slx4-S486A mutant (4.21c).
Together, these results demonstrate that the reduced DNA damage checkpoint activation in the slx4-S486A mutant promotes faster repair after treatment with MMS.
4.4.2 Reduced DNA damage checkpoint activation promotes DNA repair by activating Mus81-Mms4
One possible explanation for the rescue of the slx4-S486A mutant by the DNA damage checkpoint mutants might be as follows. First, the partially inactive checkpoint might promote the activation of an alternative pathway to resolve X-shaped DNA structures in the slx4-S486A mutant. Alternatively, the repair of X-shaped DNA structures may be still dependent on Mus81-Mms4 allowing the Slx4-Dpb11-Mms4-Mus81 complex formation even in the slx4-S486A mutant background.
To ascertain whether the rescue of the slx4-S486A mutant sensitivity to MMS by the DNA damage checkpoint mutants depends on Mus81-Mms4, we analyzed the slx4-S486A ddc1-T602A mutant sensitivity to MMS in the absence of Mms4.
Interestingly, we observed that after deleting MMS4 in the of slx4-S486A ddc1-T602A background, the cells became more sensitive to MMS than the slx4-S486A ddc1-T602A mutant (Figure 4.22a). Furthermore, after MMS treatment in S-phase, the mms4Δ deletion mutant was not able to recover after 3 hours as judged from the Rad53 phosphorylation status in the mms4Δ deletion mutant. Even the partial
inactivation of the DNA damage checkpoint did not promote faster recovery of the mms4Δ deletion mutant. In the mms4Δ ddc1-T602A mutant Rad53 remained phosphorylated during 3 hours of recovery similar to the mms4Δ mutant (Figure 4.22b). This confirms the hypothesis that the repair of X-shaped DNA structures in the slx4-S486A mutant is dependent on Mus81-Mms4.
Figure 4.22. The DNA damage checkpoint promotes DNA repair by Mus81-Mms4 but not Sgs1 in the slx4-S486A mutant after MMS damage.
a) MMS sensitivity of WT and the slx4-S486A, ddc1-T602A, mms4Δ deletion mutants and double and triple mutant combinations. Cells were spotted in serial dilutions on plates containing MMS. The growth was evaluated after incubation for 2 days at 30°C; b) Recovery experiment of WT, the mms4Δ deletion, ddc1-T602A and mms4Δ ddc1-T602A mutants. Cells were synchronized in G1 and released into S-phase in medium containing MMS. After 30 min cells were released to drug-free medium for 3 hours. The samples were taken at different time points and DNA damage checkpoint activation was evaluated by Western blot using antibodies against Rad53; c) MMS sensitivity of WT and the slx4-S486A, ddc1-T602A, sgs1Δ deletion mutants and double and triple mutant combinations. Cells were spotted as in a.
Next, we also tested whether alternative dissolution pathway is important for the rescue of the slx4-S486A mutant when the DNA damage checkpoint is partially
WT
slx4-S486A mms4Δ
slx4-S486A mms4Δ ddc1-T602A mms4Δ ddc1-T602A slx4-S486A ddc1-T602A slx4-S486A mms4Δ ddc1-T602A
0.01% MMS 0.03% MMS
- MMS
+MMS G12h 1h
3h2h
2h G1 1h
+MMS Recovery Recovery Recovery
+MMS
G1 1h 3h
WT mms4Δ
+MMS
G1 1h 3h
ddc1-T602A mms4Δ ddc1-T602A
Rad53 anti-Rad53
Recovery
3h2h
WT
slx4-S486A sgs1Δ
slx4-S486A sgs1Δ ddc1-T602A sgs1Δ ddc1-T602A slx4-S486A ddc1-T602A slx4-S486A sgs1Δ ddc1-T602A
0.01% MMS 0.025% MMS - MMS
a)
b)
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inactive. For this purpose, SGS1 was deleted in the slx4-S486A ddc1-T602A mutant and tested for the growth on MMS. In contrast to previous experiment when MMS4 was deleted, in the absence of SGS1 reduced DNA damage checkpoint activation was able to rescue the slx4-S486A mutant sensitivity to MMS (Figure 4.22c). Therefore we conclude that an alternative dissolution pathway is not important for the repair of X-shaped DNA structures in the slx4-S486A ddc1-T602A mutant.
Figure 4.23. The DNA damage checkpoint regulates the formation of the Slx4-Dpb11-Mms4-Mus81 complex.
Recovery experiment of WT, the slx4-S486A, ddc1-T602A and slx4-S486A ddc1-T602A mutants. Cells were synchronized in G1, released into S-phase in medium containing MMS.
After 30 min cells were released to drug-free medium for 2 hours. The samples were taken at different time points and evaluated by Western blot using antibodies against Rad53.
A partially inactive DNA damage checkpoint is beneficial in the slx4-S486A mutant because it promotes Mus81-Mms4-dependent DNA repair. To investigate how this is achieved mechanistically, we followed the Slx4-Dpb11-Mms4-Mus81 complex formation after a pulse of MMS damage. As we observed in our previous experiments, Mus81-Mms4 interacts with Dpb11-Slx4 after phosphorylation of Mms4 by the Polo-like kinase Cdc5 in mitosis (Figure 4.17). Interestingly, after recovery from MMS damage in S-phase Mms4 phosphorylation in the slx4-S486A mutant was delayed for 30 minutes compared with the wild type (Figure 4.23, lanes 3 and 8).
Strikingly, partial inactivation of the DNA damage checkpoint allowed Mms4 phosphorylation to occur earlier in the slx4-S486A mutant (Figure 4.23, lanes 8 and 13). After one hour of recovery Mms4 was phosphorylated in either WT or the slx4-S486A ddc1-T602A mutant (Figure 4.23, lanes 3 and 13). Moreover, Mms4 phosphorylation was consistent with Cdc5 expression. Interestingly, phosphorylation of Mms4 inversely correlated with the activation of DNA damage checkpoint. At the
0.5h 1.5h 1h 1.5h
+MMS 2h 0.5h
2h
1h 0.5h 1.5h +MMS 2h1h
Rad53 Mms43FLAG anti-FLAG
anti-Rad53
+MMS Recovery Recovery Recovery WT slx4-S486A
slx4-S486A ddc1-T602A
anti-Cdc5
Cdc5
time point when Rad53 was phosphorylated there was no phosphorylation of Mms4 and vice versa (Figure 4.23). All together, these data suggest that DNA damage checkpoint negatively regulates the formation of the Slx4-Dpb11-Mms4-Mus81 complex.