Fig 2: The RPA-binding motifs in BLM and RMI1 interact with the RPA1 N-terminal OB-fold.a BPA-modified peptide pulldowns from HeLa nuclear extracts with UV-crosslinking and denaturing washes where indicated, demonstrating that motifs 1, 3, and 5 interact directly with the RPA1 subunit of RPA. b Sequence alignments showing the similarity of RPA1-binding motifs in DNA damage response proteins. c Peptide pulldowns from rabbit reticulocyte lysates containing in vitro translated heterotrimeric RPA complexes incorporating either WT or R41E/Y42F RPA1, showing that all three BLM/RMI1 motifs cannot interact with the mutant RPA complex. d GFP-pulldowns from 293FT cells transfected with constructs expressing GFP or the indicated GFP-tagged RPA1 variants, showing that mutation of the N-terminal OB-fold of RPA1 (R41E/Y42F) disrupts binding to the BTR complex and MRE11. XPA is a negative control because it binds to RPA via different domains.

Fig 3: MYCN binds the PKMYT1 and BLM promoters in vivo. A, chromatin immunoprecipitation assays showing binding of MYCN in the region of the PKMYT1 promoter containing a putative E-box in the proximity of the transcription start site. The binding was detected in the MYCN-amplified cell lines LAN1 and NB19 but not in the non-MYCN-amplified SHSY5Y cells. B, chromatin immunoprecipitation assays showing binding of MYCN in the region of the BLM promoter containing a canonical E-box. Neg. cntr, negative control.

Fig 4: Uncoupling of BLM from Mcm6 in S-phase causes BLM-helicase-dependent acceleration of DNA replication speed. (A) DNA fiber analysis was used to compare replication speed between BLMKOcells (KSVS1452) complemented with wildtype BLM or BLM mutants that fail to bind Mcm6 in S phase (blm-WLVICI), in G1 phase (blm-QVFF), or in G1 and S-phase (blm-QVFF-FYF). Unperturbed cells were incubated in the presence of CIdU for 30 min and then in the presence of IdU for 30 min, immunostained and imaged on a Keyence fluorescence microscope. A minimum of 100 fibers showing a red region extended by a green region (elongating forks) were analyzed for each cell line, and the length (µm) of the green (IdU) region served as a measure of replication speed. A Mann-Whitney test was performed to determine statistical significance of IdU track length differences between the wildtype BLM-complemented cell line (+BLM) and the mutant BLM cell lines: **** P< 0.0001; ns, not significant. (B) DNA fiber analysis was used to compare replication speed between BLMKOcells (KSVS1452) stably expressing blm-WLVICI, blm-K695R (clone #13), or blm-WLVICI-K695R (clone #11) alleles of BLM. A minimum of 100 fibers were imaged and analyzed as in (A). (C) New origin firing was measured in BLM wildtype cells (GM00637), BLMKOcells (KSVS1452), and BLMKOcells (KSVS1452) complemented with wildtype BLM or BLM mutants that fail to bind Mcm6 in S phase (blm-WLVICI) or in G1 phase (blm-QVFF). A minimum of 200 DNA fibers from three different experiments were imaged as in (B) and the fraction of DNA fibers only stained with IdU (indicative of origin activation during the second labeling pulse) determined. A t-test was performed to determine statistical significance of differences between the BLM-complemented cell line (+BLM) and the mutant cell lines: * P< 0.05; ns, not significant. (D) Sister fork asymmetry was determined in BLM-proficient and blm-mutant cell lines by determining the ratio between the length of IdU labels on sister forks; i.e, two IdU labels extending from the same CIdU label. Between 25–40 sister forks per cell line were analyzed. Median ratio is indicated above the scatter dot blot. A Mann-Whitney test was performed to determine statistical significance of differences between the BLM-complemented cell line (+BLM) and the mutant cell lines. ** P< 0.01, **** P< 0.0001; ns, not significant.

Fig 5: BLM binds to Mcm6 in unperturbed G1 and S-phase. (A) BLM+/+(GM00637) cells were arrested at the G1/S boundary by double thymidine block, released into S-phase and BLM expression levels analyzed by Western blot. (B) Co-immunoprecipitations of endogenous BLM were performed in triplicate on nuclear extracts from BLM+/+(GM00637) cells in mid-S phase and peptides were identified by mass spectrometry. The same analysis in triplicate for the isogenic BLM knockout cell line KSVS1452 (BLMKO) yielded zero hits for the listed proteins. Cell lines GM00637 and KSVS1452 are an isogenic pair of a BLM+/+ and a CRISPR-mediated BLMKO cell line, respectively. (C, D) Reciprocal co-immunoprecipitations of endogenous BLM and Mcm6 were performed on nuclear extracts of BLM+/+ (GM00637) cells at G1/S and in mid-S phase. (E) Laser-scanning confocal microscopy images of fixed GM00637 (BLM+/+) cells and KSVS1452 (BLMKO) cells showing immunostaining of endogenous BLM [anti-BLM C18 (SCBT)] and Mcm6 [anti-Mcm6 H8 (SCBT) (F) Same cover slips of immunostained GM00637 (BLM+/+) and KSVS1452 (BLMKO) cells imaged in (E) were imaged on a Perkin Elmer ERS spinning disk confocal system visualizing large replication foci. For a different BLM and Mcm6 antibody combination and additional Mcm6 antibody verification, see Supplemental Figure S2A–C. (G) Reciprocal co-immunoprecipitations of recombinant human BLM (from yeast) and Mcm6 (from wheat germ). (H) BLM+/+ cells (GM00637) were serum-starved for 16 hours, released for 2 h, blocked at G2/M with nocodazole, released for 6 h (mid-G1) and chromatin-bound proteins extracted by biochemical fractionation. Endogenous BLM was co-immunoprecipitated from the chromatin-bound protein fractions isolated in mid-G1 and in mid-S-phase.