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TopBP1 Stabilizes BLM Protein to Suppress Sister Chromatid Exchange
Molecular Cell
Letter TopBP1 Stabilizes BLM Protein to Suppress Sister Chromatid Exchange Jiadong Wang,1 Junjie Chen,2,* and Zihua Gong2,* 1Institute of Systems Biomedicine, Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, P.R. China 2Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA *Correspondence: [email protected] (J.C.), [email protected] (Z.G.) http://dx.doi.org/10.1016/j.molcel.2015.02.011
Human TopBP1 plays essential roles in DNA replication and replication checkpoint control. In our recent paper ‘‘TopBP1 controls BLM protein level to maintain genome stability,’’ we provided evidence suggesting that TopBP1 has an unexpected role in suppressing sister chromatid exchange (SCE) and that this function is independent of its known activities in replication checkpoint control (Wang et al., 2013). In their Matters Arising (Blackford et al., 2015, this issue of Molecular Cell), Blackford et al. were able to reproduce our findings, which include the following: (1) BLM and BLM-TopBP1 interaction are dispensable for replication checkpoint signaling, (2) the interaction between TopBP1 and BLM requires the BRCT5 domain of TopBP1, (3) this interaction is phosphorylation dependent, and (4) this interaction is involved in suppression of SCEs. While we demonstrated in our study that TopBP1 suppresses SCEs via its ability to stabilize BLM in S phase cells, Blackford et al. (2015) were not able to reproduce these data. Unfortunately, they also failed to provide any new evidence or alternative mechanism to explain this function of TopBP1. In their Matters Arising, Blackford et al. (2015) questioned the role of TopBP1 in maintaining BLM protein level (Wang et al., 2013). In our study, we observed that depletion of TopBP1 downregulated BLM protein level (please see Figure 3A in Wang et al., 2013). Furthermore, we performed rescue experiments and showed that re-expression of TopBP1 was able to recover BLM protein level (please see Figure 3E in Wang et al., 2013), which clearly demonstrated that TopBP1 maintains BLM level in vivo. These important controls were ignored by Blackford et al. in their Matters Arising. We also observed that BLM protein level
was restored following MG132 treatment in TopBP1-depleted cells (please see Figure 4A in Wang et al., 2013), again supporting our conclusion that TopBP1 stabilizes BLM in S phase cells. As mentioned in the Blackford et al. summary, ‘‘we establish that disrupting the BLM-TopBP1 interaction does not markedly affect BLM stability.’’ It is possible that inefficient TopBP1 depletion may be the reason that they failed to observe a significant reduction in BLM protein level. For example, Blackford et al. (2015) in their Matters Arising used E4orf6 to downregulate TopBP1 expression. While we cannot comment on exactly how efficient TopBP1 was downregulated in their experiments, it is worthy knowing that E4orf6 has many cellular targets. Directly relevant to the issue here, there was substantial residual amount of TopBP1 in cells infected with hr703 virus (please see Figure 4 in Blackford et al., 2010, the paper they cited). This raises the possibility that the failure to observe any change in BLM protein level may again be due to inefficient TopBP1 depletion in cells infected with hr703 virus. In agreement with our report, Blackford et al. showed that the TopBP1/BLM interaction is phosphorylation dependent. However, they claimed that BLM phosphorylation on Ser304, but not on Ser338, is required for TopBP1 binding. As we showed in our manuscript, we used two independent approaches to identify the phosphorylation site required for BLM-TopBP1 interaction. We generated several BLM internal deletion mutants, performed GST pull-down experiments, and found that a small region of BLM, which was deleted in the D2 mutant, is required for its binding to TopBP1 (please see Figure 2D in Wang et al., 2013). In addition, we isolated BLM protein from G1 or S phase cells and per-
formed mass spectrometry analysis to identify potential phosphorylation sites on BLM. We found that only mutating Ser338 site, which is an S phase-specific phosphorylation site, greatly reduced the BLM/TopBP1 interaction (please see Figures 2D, S1B, and S1C in Wang et al., 2013). Furthermore, we found that the Ser338 site of BLM is phosphorylated in vivo and that this phosphorylation is cell cycle regulated (please see Figure 2G in Wang et al., 2013). To further validate our conclusion that Ser338 site is phosphorylated in vivo, we searched several large-scale phosphosite resources. We found that four other groups also reported that Ser338 is phosphorylated in vivo in human and mouse cells (Huttlin et al., 2010; Olsen et al., 2010; Wu et al., 2012; Zhou et al., 2013). We did not show phenotype for cells expressing BLM-S338. The BLM S338A mutant is unstable (please see Figure S2D in Wang et al., 2013). It is difficult to know how to select for the reconstituted cells for SCE experiments. If we select for cells with the steady-state level of S338A lower than that of wild-type BLM, we would observe the expected defect, but be criticized for using cells with lower expression level. However, if we select for cells with the steady-state level of S338A similar to that of wild-type BLM, we would expect that this mutant should fully rescue phenotypes in BLMdeficient cells, since our hypothesis is that TopBP1 only affects BLM stability, but not BLM function. We do not exclude the possibility that additional phosphorylation sites may also be involved in regulating the interaction between BLM and TopBP1. As shown in Figures 2D and S1C in Wang et al. (2013), Ser338-to-Ala mutation only reduced, but did not abolish, the interaction between BLM and TopBP1. There
Molecular Cell 57, March 19, 2015 ª2015 Elsevier Inc. 955
Molecular Cell
Letter are at least 13 phosphorylation sites on BLM that we identified using mass spectrometry analysis (please see Figure S1B in Wang et al., 2013). Therefore, it is not surprising that Blackford et al. were able to identify yet another phosphorylation site on BLM. However, the S304 phosphorylation of BLM mapped by Blackford et al. is not cell cycle regulated or induced by DNA damage. Blackford et al. also claimed that MDC1 binding to TopBP1 did not depend on BRCT domain 5 as previously reported (Wang et al., 2011). As we presented in our previous manuscript (Wang et al., 2011), we were interested in identifying BRCT5-associated proteins for many years. We showed 12 years ago that the BRCT5 domain, but not the other BRCT domains, is involved in the regulation of TopBP1 localization following DNA damage (Yamane et al., 2002). We took an unbiased approach and performed tandem affinity purification using stable cell lines expressing the SFB-tagged BRCT5 of TopBP1 and discovered MDC1 as a major TopBP1 BRCT5-associated protein in chromatin fraction (Wang et al., 2011). We provided a series of experiments to support an interaction between MDC1 and the BRCT5 domain of TopBP1 (Wang et al., 2011). In addition, structural analysis further supported this interaction between the TopBP1 BRCT5 domain and phosphorylated MDC1 (Leung et al., 2013). Interestingly, we found that MDC1 and BLM are, respectively, the major TopBP1 BRCT5 domain-associated proteins in chromatin and soluble fractions (please see Figure 1A in Wang et al., 2013), which was the starting point of our story. Based on our findings, we propose that TopBP1 BRCT5 domain may bind to different partners in soluble or chromatin fractions and therefore
carry out distinct functions (please see Figure 5H in Wang et al., 2013). Blackford et al. argued that since the K3A mutant (K38A/K39A/K40A) of BLM was expressed at levels similar to those of wild-type BLM or lysines 38, 39, or 40 were unlikely to control BLM turnover. Again, our model suggests that TopBP1 stabilizes BLM in S phase cells. BLM only became unstable in S phase cells when TopBP1 was depleted. Therefore, one would not expect to observe a drastic difference in the expression of wild-type or the K3A mutant of BLM in cells expressing endogenous TopBP1. We showed that BLM was not protected by TopBP1 in G1 cells and therefore that BLM was less stable in G1 (please see Figure S2B in Wang et al., 2013). Accordingly, we showed that the K3A mutant of BLM was more stable than wild-type BLM only in G1 cells (please see Figure S2C in Wang et al., 2013). These data agree with our hypothesis that BLM is normally degraded in G1 cells, but stabilized by TopBP1 in S phase cells. Blackford et al. showed that the BLM-K3A mutant also abolished the interaction between BLM and TOP3A/RMI2. Since we have not yet checked these interactions, we cannot comment on this observation. We did show that the K3A mutant of BLM dramatically decreased MIB1-mediated BLM ubiquitination (please see Figure 4F in Wang et al., 2013). Depletion of MIB1 restored BLM protein level in TopBP1depleted cells (please see Figure 5C in Wang et al., 2013). In addition, while depletion of TopBP1 increased SCE, codepletion of MIB1 reversed this phenotype (please see Figure 5D in Wang et al., 2013). These data suggested that MIB1 is an E3 ligase that counteracts with TopBP1 in BLM regulation.
956 Molecular Cell 57, March 19, 2015 ª2015 Elsevier Inc.
Blackford et al. also argued that BLM overexpression alone would be unlikely to override the requirement for CDKdependent phosphorylation of CtIP and NBS1, which are needed for effective resection but do not occur in G1 cells. However, besides CtIP and MRN complex, there are several other nucleases that are known to be involved in DNA end resection. It is possible that BLM overexpression may facilitate the functions of other nucleases, such as DNA2 or EXO1, and therefore lead to aberration end resection in G1 cells. REFERENCES Blackford, A.N., Patel, R.N., Forrester, N.A., Theil, K., Groitl, P., Stewart, G.S., Taylor, A.M., Morgan, I.M., Dobner, T., Grand, R.J., and Turnell, A.S. (2010). Proc. Natl. Acad. Sci. USA 107, 12251– 12256. Blackford, A.N., Nieminuszczy, J., Schwab, R.A., Galanty, Y., Jackson, S.P., and Niedzwiedz, W. (2015). Mol. Cell 57, this issue, 1133–1141. Huttlin, E.L., Jedrychowski, M.P., Elias, J.E., Goswami, T., Rad, R., Beausoleil, S.A., Ville´n, J., Haas, W., Sowa, M.E., and Gygi, S.P. (2010). Cell 143, 1174–1189. Leung, C.C., Sun, L., Gong, Z., Burkat, M., Edwards, R., Assmus, M., Chen, J., and Glover, J.N. (2013). Structure 21, 1450–1459. Olsen, J.V., Vermeulen, M., Santamaria, A., Kumar, C., Miller, M.L., Jensen, L.J., Gnad, F., Cox, J., Jensen, T.S., Nigg, E.A., et al. (2010). Sci. Signal. 3, ra3. Wang, J., Gong, Z., and Chen, J. (2011). J. Cell Biol. 193, 267–273. Wang, J., Chen, J., and Gong, Z. (2013). Mol. Cell 52, 667–678. Wu, X., Tian, L., Li, J., Zhang, Y., Han, V., Li, Y., Xu, X., Li, H., Chen, X., Chen, J., et al. (2012). Mol. Cell. Proteomics 11, 1640–1651. Yamane, K., Wu, X., and Chen, J. (2002). Mol. Cell. Biol. 22, 555–566. Zhou, H., Di Palma, S., Preisinger, C., Peng, M., Polat, A.N., Heck, A.J., and Mohammed, S. (2013). J. Proteome Res. 12, 260–271.
Letter TopBP1 Stabilizes BLM Protein to Suppress Sister Chromatid Exchange Jiadong Wang,1 Junjie Chen,2,* and Zihua Gong2,* 1Institute of Systems Biomedicine, Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, P.R. China 2Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA *Correspondence: [email protected] (J.C.), [email protected] (Z.G.) http://dx.doi.org/10.1016/j.molcel.2015.02.011
Human TopBP1 plays essential roles in DNA replication and replication checkpoint control. In our recent paper ‘‘TopBP1 controls BLM protein level to maintain genome stability,’’ we provided evidence suggesting that TopBP1 has an unexpected role in suppressing sister chromatid exchange (SCE) and that this function is independent of its known activities in replication checkpoint control (Wang et al., 2013). In their Matters Arising (Blackford et al., 2015, this issue of Molecular Cell), Blackford et al. were able to reproduce our findings, which include the following: (1) BLM and BLM-TopBP1 interaction are dispensable for replication checkpoint signaling, (2) the interaction between TopBP1 and BLM requires the BRCT5 domain of TopBP1, (3) this interaction is phosphorylation dependent, and (4) this interaction is involved in suppression of SCEs. While we demonstrated in our study that TopBP1 suppresses SCEs via its ability to stabilize BLM in S phase cells, Blackford et al. (2015) were not able to reproduce these data. Unfortunately, they also failed to provide any new evidence or alternative mechanism to explain this function of TopBP1. In their Matters Arising, Blackford et al. (2015) questioned the role of TopBP1 in maintaining BLM protein level (Wang et al., 2013). In our study, we observed that depletion of TopBP1 downregulated BLM protein level (please see Figure 3A in Wang et al., 2013). Furthermore, we performed rescue experiments and showed that re-expression of TopBP1 was able to recover BLM protein level (please see Figure 3E in Wang et al., 2013), which clearly demonstrated that TopBP1 maintains BLM level in vivo. These important controls were ignored by Blackford et al. in their Matters Arising. We also observed that BLM protein level
was restored following MG132 treatment in TopBP1-depleted cells (please see Figure 4A in Wang et al., 2013), again supporting our conclusion that TopBP1 stabilizes BLM in S phase cells. As mentioned in the Blackford et al. summary, ‘‘we establish that disrupting the BLM-TopBP1 interaction does not markedly affect BLM stability.’’ It is possible that inefficient TopBP1 depletion may be the reason that they failed to observe a significant reduction in BLM protein level. For example, Blackford et al. (2015) in their Matters Arising used E4orf6 to downregulate TopBP1 expression. While we cannot comment on exactly how efficient TopBP1 was downregulated in their experiments, it is worthy knowing that E4orf6 has many cellular targets. Directly relevant to the issue here, there was substantial residual amount of TopBP1 in cells infected with hr703 virus (please see Figure 4 in Blackford et al., 2010, the paper they cited). This raises the possibility that the failure to observe any change in BLM protein level may again be due to inefficient TopBP1 depletion in cells infected with hr703 virus. In agreement with our report, Blackford et al. showed that the TopBP1/BLM interaction is phosphorylation dependent. However, they claimed that BLM phosphorylation on Ser304, but not on Ser338, is required for TopBP1 binding. As we showed in our manuscript, we used two independent approaches to identify the phosphorylation site required for BLM-TopBP1 interaction. We generated several BLM internal deletion mutants, performed GST pull-down experiments, and found that a small region of BLM, which was deleted in the D2 mutant, is required for its binding to TopBP1 (please see Figure 2D in Wang et al., 2013). In addition, we isolated BLM protein from G1 or S phase cells and per-
formed mass spectrometry analysis to identify potential phosphorylation sites on BLM. We found that only mutating Ser338 site, which is an S phase-specific phosphorylation site, greatly reduced the BLM/TopBP1 interaction (please see Figures 2D, S1B, and S1C in Wang et al., 2013). Furthermore, we found that the Ser338 site of BLM is phosphorylated in vivo and that this phosphorylation is cell cycle regulated (please see Figure 2G in Wang et al., 2013). To further validate our conclusion that Ser338 site is phosphorylated in vivo, we searched several large-scale phosphosite resources. We found that four other groups also reported that Ser338 is phosphorylated in vivo in human and mouse cells (Huttlin et al., 2010; Olsen et al., 2010; Wu et al., 2012; Zhou et al., 2013). We did not show phenotype for cells expressing BLM-S338. The BLM S338A mutant is unstable (please see Figure S2D in Wang et al., 2013). It is difficult to know how to select for the reconstituted cells for SCE experiments. If we select for cells with the steady-state level of S338A lower than that of wild-type BLM, we would observe the expected defect, but be criticized for using cells with lower expression level. However, if we select for cells with the steady-state level of S338A similar to that of wild-type BLM, we would expect that this mutant should fully rescue phenotypes in BLMdeficient cells, since our hypothesis is that TopBP1 only affects BLM stability, but not BLM function. We do not exclude the possibility that additional phosphorylation sites may also be involved in regulating the interaction between BLM and TopBP1. As shown in Figures 2D and S1C in Wang et al. (2013), Ser338-to-Ala mutation only reduced, but did not abolish, the interaction between BLM and TopBP1. There
Molecular Cell 57, March 19, 2015 ª2015 Elsevier Inc. 955
Molecular Cell
Letter are at least 13 phosphorylation sites on BLM that we identified using mass spectrometry analysis (please see Figure S1B in Wang et al., 2013). Therefore, it is not surprising that Blackford et al. were able to identify yet another phosphorylation site on BLM. However, the S304 phosphorylation of BLM mapped by Blackford et al. is not cell cycle regulated or induced by DNA damage. Blackford et al. also claimed that MDC1 binding to TopBP1 did not depend on BRCT domain 5 as previously reported (Wang et al., 2011). As we presented in our previous manuscript (Wang et al., 2011), we were interested in identifying BRCT5-associated proteins for many years. We showed 12 years ago that the BRCT5 domain, but not the other BRCT domains, is involved in the regulation of TopBP1 localization following DNA damage (Yamane et al., 2002). We took an unbiased approach and performed tandem affinity purification using stable cell lines expressing the SFB-tagged BRCT5 of TopBP1 and discovered MDC1 as a major TopBP1 BRCT5-associated protein in chromatin fraction (Wang et al., 2011). We provided a series of experiments to support an interaction between MDC1 and the BRCT5 domain of TopBP1 (Wang et al., 2011). In addition, structural analysis further supported this interaction between the TopBP1 BRCT5 domain and phosphorylated MDC1 (Leung et al., 2013). Interestingly, we found that MDC1 and BLM are, respectively, the major TopBP1 BRCT5 domain-associated proteins in chromatin and soluble fractions (please see Figure 1A in Wang et al., 2013), which was the starting point of our story. Based on our findings, we propose that TopBP1 BRCT5 domain may bind to different partners in soluble or chromatin fractions and therefore
carry out distinct functions (please see Figure 5H in Wang et al., 2013). Blackford et al. argued that since the K3A mutant (K38A/K39A/K40A) of BLM was expressed at levels similar to those of wild-type BLM or lysines 38, 39, or 40 were unlikely to control BLM turnover. Again, our model suggests that TopBP1 stabilizes BLM in S phase cells. BLM only became unstable in S phase cells when TopBP1 was depleted. Therefore, one would not expect to observe a drastic difference in the expression of wild-type or the K3A mutant of BLM in cells expressing endogenous TopBP1. We showed that BLM was not protected by TopBP1 in G1 cells and therefore that BLM was less stable in G1 (please see Figure S2B in Wang et al., 2013). Accordingly, we showed that the K3A mutant of BLM was more stable than wild-type BLM only in G1 cells (please see Figure S2C in Wang et al., 2013). These data agree with our hypothesis that BLM is normally degraded in G1 cells, but stabilized by TopBP1 in S phase cells. Blackford et al. showed that the BLM-K3A mutant also abolished the interaction between BLM and TOP3A/RMI2. Since we have not yet checked these interactions, we cannot comment on this observation. We did show that the K3A mutant of BLM dramatically decreased MIB1-mediated BLM ubiquitination (please see Figure 4F in Wang et al., 2013). Depletion of MIB1 restored BLM protein level in TopBP1depleted cells (please see Figure 5C in Wang et al., 2013). In addition, while depletion of TopBP1 increased SCE, codepletion of MIB1 reversed this phenotype (please see Figure 5D in Wang et al., 2013). These data suggested that MIB1 is an E3 ligase that counteracts with TopBP1 in BLM regulation.
956 Molecular Cell 57, March 19, 2015 ª2015 Elsevier Inc.
Blackford et al. also argued that BLM overexpression alone would be unlikely to override the requirement for CDKdependent phosphorylation of CtIP and NBS1, which are needed for effective resection but do not occur in G1 cells. However, besides CtIP and MRN complex, there are several other nucleases that are known to be involved in DNA end resection. It is possible that BLM overexpression may facilitate the functions of other nucleases, such as DNA2 or EXO1, and therefore lead to aberration end resection in G1 cells. REFERENCES Blackford, A.N., Patel, R.N., Forrester, N.A., Theil, K., Groitl, P., Stewart, G.S., Taylor, A.M., Morgan, I.M., Dobner, T., Grand, R.J., and Turnell, A.S. (2010). Proc. Natl. Acad. Sci. USA 107, 12251– 12256. Blackford, A.N., Nieminuszczy, J., Schwab, R.A., Galanty, Y., Jackson, S.P., and Niedzwiedz, W. (2015). Mol. Cell 57, this issue, 1133–1141. Huttlin, E.L., Jedrychowski, M.P., Elias, J.E., Goswami, T., Rad, R., Beausoleil, S.A., Ville´n, J., Haas, W., Sowa, M.E., and Gygi, S.P. (2010). Cell 143, 1174–1189. Leung, C.C., Sun, L., Gong, Z., Burkat, M., Edwards, R., Assmus, M., Chen, J., and Glover, J.N. (2013). Structure 21, 1450–1459. Olsen, J.V., Vermeulen, M., Santamaria, A., Kumar, C., Miller, M.L., Jensen, L.J., Gnad, F., Cox, J., Jensen, T.S., Nigg, E.A., et al. (2010). Sci. Signal. 3, ra3. Wang, J., Gong, Z., and Chen, J. (2011). J. Cell Biol. 193, 267–273. Wang, J., Chen, J., and Gong, Z. (2013). Mol. Cell 52, 667–678. Wu, X., Tian, L., Li, J., Zhang, Y., Han, V., Li, Y., Xu, X., Li, H., Chen, X., Chen, J., et al. (2012). Mol. Cell. Proteomics 11, 1640–1651. Yamane, K., Wu, X., and Chen, J. (2002). Mol. Cell. Biol. 22, 555–566. Zhou, H., Di Palma, S., Preisinger, C., Peng, M., Polat, A.N., Heck, A.J., and Mohammed, S. (2013). J. Proteome Res. 12, 260–271.