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.