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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.

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The BLM-TopBP1 Interaction Promotes Genome Stability and Requires Ser304 but Not Ser338 of BLM(A) Mutation of BLM Ser338 to alanine does not affect its interaction with endogenous TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(B) Mutation of BLM Ser338 to alanine does not affect its interaction with recombinant GST-tagged TopBP1-BRCT5. Pull-downs were carried out using GST proteins bound to glutathione beads incubated with lysates from 293FT cells transiently transfected with the indicated plasmids.(C) TopBP1-BRCT5 interacts directly with BLM peptides encompassing phosphorylated Ser304 but not Ser338. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT5 or GST alone.(D) Analysis of SCEs in U2OS cells depleted of endogenous TopBP1 with siRNAs targeting the 3′ UTR and expressing wild-type or K704E TopBP1. A minimum of 20 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test.(E) Analysis of SCEs in DT40 cells. A minimum of 50 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test. “cl.,” clone.(F) DNA fiber analyses to measure origin firing. DT40 cells were treated with 2.5 μM camptothecin for 90 min in the presence of IdU, washed, and then released into drug-free medium containing CldU for 15 min. A minimum of 200 fibers were scored per experiment. Mean values of three independent experiments are shown ± SEM. Significance was determined using Student’s two-tailed t test.(G) Analysis of chromosomal aberrations. Mitotic spreads were prepared from DT40 cells treated with 2 μM aphidicolin for 12 hr. A minimum of 35 metaphases was scored per experiment. Significance was determined using Mann-Whitney U test. See also Figure S3.
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fig3: The BLM-TopBP1 Interaction Promotes Genome Stability and Requires Ser304 but Not Ser338 of BLM(A) Mutation of BLM Ser338 to alanine does not affect its interaction with endogenous TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(B) Mutation of BLM Ser338 to alanine does not affect its interaction with recombinant GST-tagged TopBP1-BRCT5. Pull-downs were carried out using GST proteins bound to glutathione beads incubated with lysates from 293FT cells transiently transfected with the indicated plasmids.(C) TopBP1-BRCT5 interacts directly with BLM peptides encompassing phosphorylated Ser304 but not Ser338. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT5 or GST alone.(D) Analysis of SCEs in U2OS cells depleted of endogenous TopBP1 with siRNAs targeting the 3′ UTR and expressing wild-type or K704E TopBP1. A minimum of 20 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test.(E) Analysis of SCEs in DT40 cells. A minimum of 50 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test. “cl.,” clone.(F) DNA fiber analyses to measure origin firing. DT40 cells were treated with 2.5 μM camptothecin for 90 min in the presence of IdU, washed, and then released into drug-free medium containing CldU for 15 min. A minimum of 200 fibers were scored per experiment. Mean values of three independent experiments are shown ± SEM. Significance was determined using Student’s two-tailed t test.(G) Analysis of chromosomal aberrations. Mitotic spreads were prepared from DT40 cells treated with 2 μM aphidicolin for 12 hr. A minimum of 35 metaphases was scored per experiment. Significance was determined using Mann-Whitney U test. See also Figure S3.

Mentions: Our conclusion that Ser304 is the critical residue of BLM that interacts with TopBP1 BRCT domain 5 differed from that of a recent report describing a role for Ser338 in this binding (Wang et al., 2013). We initially considered that our respective studies were not necessarily in disagreement, since it might have been that phosphorylation of multiple residues was required for the BLM-TopBP1 interaction. We therefore tested whether we could replicate a role for BLM-Ser338 in mediating TopBP1 binding. To do this, we expressed GFP-tagged wild-type BLM or derivatives in which Ser304 or Ser338 was mutated to alanine (S304A and S338A, respectively) in cells and assessed their abilities to retrieve endogenous TopBP1 from cell extracts (Figure 3A). In contrast to BLM-S304A, the S338A mutant behaved as wild-type in its ability to bind TopBP1, indicating that Ser338 is not required for the BLM-TopBP1 interaction. Analyses using cells synchronized in S phase produced similar results (Figure S3A).


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)

The BLM-TopBP1 Interaction Promotes Genome Stability and Requires Ser304 but Not Ser338 of BLM(A) Mutation of BLM Ser338 to alanine does not affect its interaction with endogenous TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(B) Mutation of BLM Ser338 to alanine does not affect its interaction with recombinant GST-tagged TopBP1-BRCT5. Pull-downs were carried out using GST proteins bound to glutathione beads incubated with lysates from 293FT cells transiently transfected with the indicated plasmids.(C) TopBP1-BRCT5 interacts directly with BLM peptides encompassing phosphorylated Ser304 but not Ser338. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT5 or GST alone.(D) Analysis of SCEs in U2OS cells depleted of endogenous TopBP1 with siRNAs targeting the 3′ UTR and expressing wild-type or K704E TopBP1. A minimum of 20 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test.(E) Analysis of SCEs in DT40 cells. A minimum of 50 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test. “cl.,” clone.(F) DNA fiber analyses to measure origin firing. DT40 cells were treated with 2.5 μM camptothecin for 90 min in the presence of IdU, washed, and then released into drug-free medium containing CldU for 15 min. A minimum of 200 fibers were scored per experiment. Mean values of three independent experiments are shown ± SEM. Significance was determined using Student’s two-tailed t test.(G) Analysis of chromosomal aberrations. Mitotic spreads were prepared from DT40 cells treated with 2 μM aphidicolin for 12 hr. A minimum of 35 metaphases was scored per experiment. Significance was determined using Mann-Whitney U test. See also Figure S3.
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fig3: The BLM-TopBP1 Interaction Promotes Genome Stability and Requires Ser304 but Not Ser338 of BLM(A) Mutation of BLM Ser338 to alanine does not affect its interaction with endogenous TopBP1. Pull-downs were carried out from 293FT cells transiently transfected with the indicated plasmids.(B) Mutation of BLM Ser338 to alanine does not affect its interaction with recombinant GST-tagged TopBP1-BRCT5. Pull-downs were carried out using GST proteins bound to glutathione beads incubated with lysates from 293FT cells transiently transfected with the indicated plasmids.(C) TopBP1-BRCT5 interacts directly with BLM peptides encompassing phosphorylated Ser304 but not Ser338. Streptavidin beads were incubated with biotinylated peptides before mixing with GST-tagged BRCT5 or GST alone.(D) Analysis of SCEs in U2OS cells depleted of endogenous TopBP1 with siRNAs targeting the 3′ UTR and expressing wild-type or K704E TopBP1. A minimum of 20 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test.(E) Analysis of SCEs in DT40 cells. A minimum of 50 metaphases was scored per experiment. Significance was determined using the Mann-Whitney U test. “cl.,” clone.(F) DNA fiber analyses to measure origin firing. DT40 cells were treated with 2.5 μM camptothecin for 90 min in the presence of IdU, washed, and then released into drug-free medium containing CldU for 15 min. A minimum of 200 fibers were scored per experiment. Mean values of three independent experiments are shown ± SEM. Significance was determined using Student’s two-tailed t test.(G) Analysis of chromosomal aberrations. Mitotic spreads were prepared from DT40 cells treated with 2 μM aphidicolin for 12 hr. A minimum of 35 metaphases was scored per experiment. Significance was determined using Mann-Whitney U test. See also Figure S3.
Mentions: Our conclusion that Ser304 is the critical residue of BLM that interacts with TopBP1 BRCT domain 5 differed from that of a recent report describing a role for Ser338 in this binding (Wang et al., 2013). We initially considered that our respective studies were not necessarily in disagreement, since it might have been that phosphorylation of multiple residues was required for the BLM-TopBP1 interaction. We therefore tested whether we could replicate a role for BLM-Ser338 in mediating TopBP1 binding. To do this, we expressed GFP-tagged wild-type BLM or derivatives in which Ser304 or Ser338 was mutated to alanine (S304A and S338A, respectively) in cells and assessed their abilities to retrieve endogenous TopBP1 from cell extracts (Figure 3A). In contrast to BLM-S304A, the S338A mutant behaved as wild-type in its ability to bind TopBP1, indicating that Ser338 is not required for the BLM-TopBP1 interaction. Analyses using cells synchronized in S phase produced similar results (Figure S3A).

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