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TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation.

Blackford AN, Nieminuszczy J, Schwab RA, Galanty Y, Jackson SP, Niedzwiedz W - Mol. Cell (2015)

Bottom Line: Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304.Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability.Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.

View Article: PubMed Central - PubMed

Affiliation: The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK; The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK.

No MeSH data available.


Related in: MedlinePlus

TopBP1 Does Not Protect BLM from Degradation(A) TopBP1 depletion does not affect BLM levels. HeLa cells were transfected with the indicated siRNAs and harvested for western blotting 3 days later.(B) Adenovirus-induced proteasomal degradation of TopBP1 does not affect BLM levels. U2OS cells were infected with the hr703 adenovirus and harvested at the indicated times for western blotting. E1A is a control for adenovirus infection.(C) BLM-TopBP1 interaction does not maintain BLM stability. Cycloheximide (CHX) was added to U2OS cells stably expressing GFP-BLM proteins for the indicated times before harvesting for western blotting. The graph shows the level of BLM at the time points indicated as a percentage of untreated (0 h). Quantification was performed using ImageJ.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Lys38, Lys39, and Lys40.(E) Mutation of BLM Lys38, Lys39, and Lys40 to alanine (K3A) disrupts binding to TOP3A and RMI2. 293FT cells were transfected with the indicated plasmids and harvested for pull-downs 24 hr later. See also Figure S4.
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fig4: TopBP1 Does Not Protect BLM from Degradation(A) TopBP1 depletion does not affect BLM levels. HeLa cells were transfected with the indicated siRNAs and harvested for western blotting 3 days later.(B) Adenovirus-induced proteasomal degradation of TopBP1 does not affect BLM levels. U2OS cells were infected with the hr703 adenovirus and harvested at the indicated times for western blotting. E1A is a control for adenovirus infection.(C) BLM-TopBP1 interaction does not maintain BLM stability. Cycloheximide (CHX) was added to U2OS cells stably expressing GFP-BLM proteins for the indicated times before harvesting for western blotting. The graph shows the level of BLM at the time points indicated as a percentage of untreated (0 h). Quantification was performed using ImageJ.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Lys38, Lys39, and Lys40.(E) Mutation of BLM Lys38, Lys39, and Lys40 to alanine (K3A) disrupts binding to TOP3A and RMI2. 293FT cells were transfected with the indicated plasmids and harvested for pull-downs 24 hr later. See also Figure S4.

Mentions: It was suggested that BLM interaction with TopBP1 is required to prevent ubiquitylation and subsequent proteasomal degradation of BLM (Wang et al., 2013). We therefore assessed whether we could also observe BLM destabilization in absence of TopBP1. To do this, we first verified that our BLM antibody was specific by showing that it recognized a protein species that is absent in Bloom syndrome cells and in cells treated with an siRNA targeting the BLM mRNA (Figures S4A and S4B). We then transfected cells with TopBP1 siRNAs with sequences that were identical to those used by Wang et al., as well as two additional ones of our own design. Strikingly, all four siRNAs depleted TopBP1 to near-undetectable levels in HeLa cells but had no appreciable effect on BLM protein stability (Figure 4A). Similar results were obtained in U2OS cells (data not shown), suggesting that TopBP1 plays no discernible role in regulating BLM levels. To seek to confirm this, we used an alternative method to reduce TopBP1 expression in cells. Some human adenoviruses promote TopBP1 degradation in infected cells by hijacking a Cullin-based E3 ubiquitin ligase with the viral E4orf6 protein and directing it toward TopBP1 (Blackford et al., 2010). We therefore used one such virus, hr703, to examine BLM levels during infection. Although TopBP1 was rapidly and almost completely degraded in cells infected with hr703, BLM levels actually increased slightly (Figure 4B). Finally, we compared the half-lives of wild-type and S304A BLM in cells treated with the translation inhibitor, cycloheximide. Ensuing results demonstrated that the half-lives of both proteins were roughly equivalent, with the S304A mutant in fact being slightly more stable (Figure 4C). Synchronization of cells in S phase yielded similar results (Figure S4C). Based on these experiments, we concluded that TopBP1 plays no significant role in controlling BLM protein levels.


TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation.

Blackford AN, Nieminuszczy J, Schwab RA, Galanty Y, Jackson SP, Niedzwiedz W - Mol. Cell (2015)

TopBP1 Does Not Protect BLM from Degradation(A) TopBP1 depletion does not affect BLM levels. HeLa cells were transfected with the indicated siRNAs and harvested for western blotting 3 days later.(B) Adenovirus-induced proteasomal degradation of TopBP1 does not affect BLM levels. U2OS cells were infected with the hr703 adenovirus and harvested at the indicated times for western blotting. E1A is a control for adenovirus infection.(C) BLM-TopBP1 interaction does not maintain BLM stability. Cycloheximide (CHX) was added to U2OS cells stably expressing GFP-BLM proteins for the indicated times before harvesting for western blotting. The graph shows the level of BLM at the time points indicated as a percentage of untreated (0 h). Quantification was performed using ImageJ.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Lys38, Lys39, and Lys40.(E) Mutation of BLM Lys38, Lys39, and Lys40 to alanine (K3A) disrupts binding to TOP3A and RMI2. 293FT cells were transfected with the indicated plasmids and harvested for pull-downs 24 hr later. See also Figure S4.
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fig4: TopBP1 Does Not Protect BLM from Degradation(A) TopBP1 depletion does not affect BLM levels. HeLa cells were transfected with the indicated siRNAs and harvested for western blotting 3 days later.(B) Adenovirus-induced proteasomal degradation of TopBP1 does not affect BLM levels. U2OS cells were infected with the hr703 adenovirus and harvested at the indicated times for western blotting. E1A is a control for adenovirus infection.(C) BLM-TopBP1 interaction does not maintain BLM stability. Cycloheximide (CHX) was added to U2OS cells stably expressing GFP-BLM proteins for the indicated times before harvesting for western blotting. The graph shows the level of BLM at the time points indicated as a percentage of untreated (0 h). Quantification was performed using ImageJ.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Lys38, Lys39, and Lys40.(E) Mutation of BLM Lys38, Lys39, and Lys40 to alanine (K3A) disrupts binding to TOP3A and RMI2. 293FT cells were transfected with the indicated plasmids and harvested for pull-downs 24 hr later. See also Figure S4.
Mentions: It was suggested that BLM interaction with TopBP1 is required to prevent ubiquitylation and subsequent proteasomal degradation of BLM (Wang et al., 2013). We therefore assessed whether we could also observe BLM destabilization in absence of TopBP1. To do this, we first verified that our BLM antibody was specific by showing that it recognized a protein species that is absent in Bloom syndrome cells and in cells treated with an siRNA targeting the BLM mRNA (Figures S4A and S4B). We then transfected cells with TopBP1 siRNAs with sequences that were identical to those used by Wang et al., as well as two additional ones of our own design. Strikingly, all four siRNAs depleted TopBP1 to near-undetectable levels in HeLa cells but had no appreciable effect on BLM protein stability (Figure 4A). Similar results were obtained in U2OS cells (data not shown), suggesting that TopBP1 plays no discernible role in regulating BLM levels. To seek to confirm this, we used an alternative method to reduce TopBP1 expression in cells. Some human adenoviruses promote TopBP1 degradation in infected cells by hijacking a Cullin-based E3 ubiquitin ligase with the viral E4orf6 protein and directing it toward TopBP1 (Blackford et al., 2010). We therefore used one such virus, hr703, to examine BLM levels during infection. Although TopBP1 was rapidly and almost completely degraded in cells infected with hr703, BLM levels actually increased slightly (Figure 4B). Finally, we compared the half-lives of wild-type and S304A BLM in cells treated with the translation inhibitor, cycloheximide. Ensuing results demonstrated that the half-lives of both proteins were roughly equivalent, with the S304A mutant in fact being slightly more stable (Figure 4C). Synchronization of cells in S phase yielded similar results (Figure S4C). Based on these experiments, we concluded that TopBP1 plays no significant role in controlling BLM protein levels.

Bottom Line: Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304.Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability.Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.

View Article: PubMed Central - PubMed

Affiliation: The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK; The Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK.

No MeSH data available.


Related in: MedlinePlus