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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

BLM Ser304 Phosphorylation Mediates Direct Binding to TopBP1-BRCT5(A) Schematic of the GFP-tagged BLM constructs used in this study.(B) The N-terminal 132 residues of BLM are required for binding to TOP3A and RMI2 but not TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(C) The binding site for TopBP1 is located within residues 133–587 of BLM. RMI2 is a positive control for binding to the N terminus of BLM.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Ser304 and Ser338.(E) Mutation of Ser304 specifically abrogates binding to TopBP1. Pull-downs were carried out from U2OS cells stably expressing the indicated proteins.(F) The BLM-pS304 antibody does not recognize BLM-S304A. 293FT cells were transiently transfected with the indicated plasmids. NBS1 is a loading control.(G) Ser304 is phosphorylated in vivo. BLM immunoprecipitates from U2OS cells were mock treated or treated with lambda phosphatase (λ-PPase).(H) BLM residues 297–311 are sufficient for interaction with TopBP1 when Ser304 is phosphorylated. Streptavidin beads were incubated with biotinylated peptides before addition to HeLa nuclear extracts for pull-downs.(I) TopBP1 BRCT domains 4 and 5 interact directly with BLM peptides phosphorylated on Ser304. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT domains 4 and 5 or GST alone. See also Figure S2.
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fig2: BLM Ser304 Phosphorylation Mediates Direct Binding to TopBP1-BRCT5(A) Schematic of the GFP-tagged BLM constructs used in this study.(B) The N-terminal 132 residues of BLM are required for binding to TOP3A and RMI2 but not TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(C) The binding site for TopBP1 is located within residues 133–587 of BLM. RMI2 is a positive control for binding to the N terminus of BLM.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Ser304 and Ser338.(E) Mutation of Ser304 specifically abrogates binding to TopBP1. Pull-downs were carried out from U2OS cells stably expressing the indicated proteins.(F) The BLM-pS304 antibody does not recognize BLM-S304A. 293FT cells were transiently transfected with the indicated plasmids. NBS1 is a loading control.(G) Ser304 is phosphorylated in vivo. BLM immunoprecipitates from U2OS cells were mock treated or treated with lambda phosphatase (λ-PPase).(H) BLM residues 297–311 are sufficient for interaction with TopBP1 when Ser304 is phosphorylated. Streptavidin beads were incubated with biotinylated peptides before addition to HeLa nuclear extracts for pull-downs.(I) TopBP1 BRCT domains 4 and 5 interact directly with BLM peptides phosphorylated on Ser304. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT domains 4 and 5 or GST alone. See also Figure S2.

Mentions: To establish whether BLM itself or one of its binding partners was mediating BLM-TopBP1 binding, we took advantage of the fact that the N-terminal 132 residues of BLM are required for its interaction with the dissolvasome (Hu et al., 2001). Importantly, deleting this region led to the expected abrogation of TOP3A and RMI2 binding, but TopBP1 binding was unaffected (Figures 2A and 2B). This result indicated that TopBP1 can associate with BLM in the absence of other dissolvasome members, thereby strongly implicating BLM as the mediator of TopBP1 binding to this complex.


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)

BLM Ser304 Phosphorylation Mediates Direct Binding to TopBP1-BRCT5(A) Schematic of the GFP-tagged BLM constructs used in this study.(B) The N-terminal 132 residues of BLM are required for binding to TOP3A and RMI2 but not TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(C) The binding site for TopBP1 is located within residues 133–587 of BLM. RMI2 is a positive control for binding to the N terminus of BLM.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Ser304 and Ser338.(E) Mutation of Ser304 specifically abrogates binding to TopBP1. Pull-downs were carried out from U2OS cells stably expressing the indicated proteins.(F) The BLM-pS304 antibody does not recognize BLM-S304A. 293FT cells were transiently transfected with the indicated plasmids. NBS1 is a loading control.(G) Ser304 is phosphorylated in vivo. BLM immunoprecipitates from U2OS cells were mock treated or treated with lambda phosphatase (λ-PPase).(H) BLM residues 297–311 are sufficient for interaction with TopBP1 when Ser304 is phosphorylated. Streptavidin beads were incubated with biotinylated peptides before addition to HeLa nuclear extracts for pull-downs.(I) TopBP1 BRCT domains 4 and 5 interact directly with BLM peptides phosphorylated on Ser304. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT domains 4 and 5 or GST alone. See also Figure S2.
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fig2: BLM Ser304 Phosphorylation Mediates Direct Binding to TopBP1-BRCT5(A) Schematic of the GFP-tagged BLM constructs used in this study.(B) The N-terminal 132 residues of BLM are required for binding to TOP3A and RMI2 but not TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(C) The binding site for TopBP1 is located within residues 133–587 of BLM. RMI2 is a positive control for binding to the N terminus of BLM.(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Ser304 and Ser338.(E) Mutation of Ser304 specifically abrogates binding to TopBP1. Pull-downs were carried out from U2OS cells stably expressing the indicated proteins.(F) The BLM-pS304 antibody does not recognize BLM-S304A. 293FT cells were transiently transfected with the indicated plasmids. NBS1 is a loading control.(G) Ser304 is phosphorylated in vivo. BLM immunoprecipitates from U2OS cells were mock treated or treated with lambda phosphatase (λ-PPase).(H) BLM residues 297–311 are sufficient for interaction with TopBP1 when Ser304 is phosphorylated. Streptavidin beads were incubated with biotinylated peptides before addition to HeLa nuclear extracts for pull-downs.(I) TopBP1 BRCT domains 4 and 5 interact directly with BLM peptides phosphorylated on Ser304. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT domains 4 and 5 or GST alone. See also Figure S2.
Mentions: To establish whether BLM itself or one of its binding partners was mediating BLM-TopBP1 binding, we took advantage of the fact that the N-terminal 132 residues of BLM are required for its interaction with the dissolvasome (Hu et al., 2001). Importantly, deleting this region led to the expected abrogation of TOP3A and RMI2 binding, but TopBP1 binding was unaffected (Figures 2A and 2B). This result indicated that TopBP1 can associate with BLM in the absence of other dissolvasome members, thereby strongly implicating BLM as the mediator of TopBP1 binding to this complex.

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