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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 Interacts with TopBP1 via BRCT5(A) TopBP1 immunoprecipitates from 293FT cell extracts contain BLM.(B) BLM immunoprecipitates from 293FT cell extracts contain TopBP1.(C) Schematic showing TopBP1 BRCT domain layout. Black numbered boxes represent BRCT domains. K154, K704, and K1317 are the key phosphopeptide binding lysines in BRCT domains 1, 5, and 7, respectively. The names of known TopBP1-binding partners are shown below the BRCT domains they interact with.(D) Effect of point mutations in TopBP1 BRCT domains 1, 5, and 7 on its binding to NBS1, MDC1, FANCJ, and BLM compared to wild-type (WT). Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids 24 hr later.(E) Effect of two different point mutations in TopBP1-BRCT5 on binding to MDC1 and BLM. NBS1 and FANCJ are positive controls as they bind to TopBP1 BRCT domains 1 and 7, respectively.(F) Mutation of TopBP1-BRCT5 abrogates binding to Bloom syndrome complex members. See also Figure S1.
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fig1: BLM Interacts with TopBP1 via BRCT5(A) TopBP1 immunoprecipitates from 293FT cell extracts contain BLM.(B) BLM immunoprecipitates from 293FT cell extracts contain TopBP1.(C) Schematic showing TopBP1 BRCT domain layout. Black numbered boxes represent BRCT domains. K154, K704, and K1317 are the key phosphopeptide binding lysines in BRCT domains 1, 5, and 7, respectively. The names of known TopBP1-binding partners are shown below the BRCT domains they interact with.(D) Effect of point mutations in TopBP1 BRCT domains 1, 5, and 7 on its binding to NBS1, MDC1, FANCJ, and BLM compared to wild-type (WT). Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids 24 hr later.(E) Effect of two different point mutations in TopBP1-BRCT5 on binding to MDC1 and BLM. NBS1 and FANCJ are positive controls as they bind to TopBP1 BRCT domains 1 and 7, respectively.(F) Mutation of TopBP1-BRCT5 abrogates binding to Bloom syndrome complex members. See also Figure S1.

Mentions: We identified BLM as a candidate TopBP1 interactor by mass spectrometric analyses of TopBP1-associated proteins (see Figure S1 available online). To validate this interaction, we carried out coimmunoprecipitations from cell extracts and found that we could readily detect BLM by western blotting of TopBP1 immunoprecipitates (Figure 1A). Consistent with this, TopBP1 was detected in reciprocal BLM immunoprecipitates from cell extracts (Figure 1B), thus confirming that the two proteins likely exist in a complex together in cells.


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 Interacts with TopBP1 via BRCT5(A) TopBP1 immunoprecipitates from 293FT cell extracts contain BLM.(B) BLM immunoprecipitates from 293FT cell extracts contain TopBP1.(C) Schematic showing TopBP1 BRCT domain layout. Black numbered boxes represent BRCT domains. K154, K704, and K1317 are the key phosphopeptide binding lysines in BRCT domains 1, 5, and 7, respectively. The names of known TopBP1-binding partners are shown below the BRCT domains they interact with.(D) Effect of point mutations in TopBP1 BRCT domains 1, 5, and 7 on its binding to NBS1, MDC1, FANCJ, and BLM compared to wild-type (WT). Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids 24 hr later.(E) Effect of two different point mutations in TopBP1-BRCT5 on binding to MDC1 and BLM. NBS1 and FANCJ are positive controls as they bind to TopBP1 BRCT domains 1 and 7, respectively.(F) Mutation of TopBP1-BRCT5 abrogates binding to Bloom syndrome complex members. See also Figure S1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374139&req=5

fig1: BLM Interacts with TopBP1 via BRCT5(A) TopBP1 immunoprecipitates from 293FT cell extracts contain BLM.(B) BLM immunoprecipitates from 293FT cell extracts contain TopBP1.(C) Schematic showing TopBP1 BRCT domain layout. Black numbered boxes represent BRCT domains. K154, K704, and K1317 are the key phosphopeptide binding lysines in BRCT domains 1, 5, and 7, respectively. The names of known TopBP1-binding partners are shown below the BRCT domains they interact with.(D) Effect of point mutations in TopBP1 BRCT domains 1, 5, and 7 on its binding to NBS1, MDC1, FANCJ, and BLM compared to wild-type (WT). Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids 24 hr later.(E) Effect of two different point mutations in TopBP1-BRCT5 on binding to MDC1 and BLM. NBS1 and FANCJ are positive controls as they bind to TopBP1 BRCT domains 1 and 7, respectively.(F) Mutation of TopBP1-BRCT5 abrogates binding to Bloom syndrome complex members. See also Figure S1.
Mentions: We identified BLM as a candidate TopBP1 interactor by mass spectrometric analyses of TopBP1-associated proteins (see Figure S1 available online). To validate this interaction, we carried out coimmunoprecipitations from cell extracts and found that we could readily detect BLM by western blotting of TopBP1 immunoprecipitates (Figure 1A). Consistent with this, TopBP1 was detected in reciprocal BLM immunoprecipitates from cell extracts (Figure 1B), thus confirming that the two proteins likely exist in a complex together in cells.

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