Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core with either the P868L or G1120R substitution. We also purified a BLM K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLM P868L and BLM G1120R were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of lobe 1 of the ATPase domain. Because BLM P868L and BLM G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLM K869A K870A has diminished ATPase activity compared to wild-type BLM, weakened binding to duplex DNA structures, and less robust helicase activity. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM.

Differential Scanning Fluorimetry: Differential scanning fluorimetry (DSF) was carried out as previously described with the following modifications. WT BLM_core, BLM_core P868L, or BLM_core G1120R (5 µM) were incubated in DSF buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, and 5 mM MgCl₂) with 5X Sypro Orange (Millipore-Sigma) in 20 µL total volume for 10 minutes at room temperature. For reactions containing ADP or ATPγS, nucleotide was added to final concentration of 0.5 mM. Samples were then heated from 4 °C to 95 °C at a rate of 0.25 °C s^-1 with fluorescence measured every two seconds using the HEX filter in the C1000 Touch Thermal Cycler (Bio-Rad). Each DSF condition was done in triplicate.

DSF data was analyzed using DSFWorld (https://gestwickilab.shinyapps.io/dsfworld/). Raw fluorescence data was uploaded to the DSFWorld and data trimmed from 30 °C to 60 °C for each sample was used to determine the T_m by determining the maximum of the first derivative (dRFU) on DSFWorld. ∆T_m values for ADP and ATPγS samples was determined by subtracting the average Tm for each protein alone from the T_m values calculated for each ADP and ATPγS replicate. Significance was determined using Welch’s two-tailed t-test using the default settings on Prism Version 9.3.1. Normalized fluorescence plots were determined by normalizing data trimmed from 30 °C to 60 °C in Prism Version 9.3.1.

ATPase assay: WT BLM, BLM P868L, or BLM G1120R (5 nM) were incubated with either no DNA or dT20 serially diluted from 10 nM to 9.76 x 10^-3 nM in ATPase buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5% (v/v) glycerol, 0.1 mM DTT, 5 mM MgCl₂, 0.1 mg/mL BSA, 2 mM 2-phosphoenolpyruvate, 3 U/mL Pyruvate Kinase/Lactate Dehydrogenase, 0.2 mM NADH). BLM K869A K870A at 5 nM was incubated with no DNA or with dT₂₀ serially diluted from 100 nM to 0.98 nM in ATPase buffer. Reactions were initiated by addition of 1 mM ATP and A_{340 nm} was monitored for 1 hour at 25 °C. Assays were done in triplicate. 

DNA binding assays: Partial duplex DNA (oRC32/FAM-oAV320, Table 1) or G4 DNA (FAM-oRC96, Table 1) constructs were folded by incubating 5 µM DNA in 10 mM Tris-HCl, pH 7.5 and 100 mM KCl at 95 °C for 10 minutes and slowly cooling the sample to room temperature over several hours. DNA constructs were stored at 4 °C. Serial dilutions of BLM or BLM mutant proteins were incubated with 40 nM partial duplex DNA, G4 DNA, or FAM-dT₃₀ in 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mg/mL BSA, and 5 mM MgCl₂ for 30 minutes at room temperature. 3.3% (v/v) glycerol was added to samples, and 5 µL of each sample was loaded onto a 5% acrylamide 1.5-mm gel in TBE buffer supplemented with 100 mM KCl. Gels were pre-run at 75 V for 20 minutes before loading protein/DNA complexes and running at 75 V for 1 hour at 4 °C in 1xTBE running buffer supplemented with 100 mM KCl. Gels were imaged on the Azure c600 (Azure Biosystems). Experiments were done in triplicate.

Helicase assays: Partial dsDNA or G4-dsDNA constructs were folded by incubating 5 µM DNA in 10 mM Tris-HCl, pH 7.5 and 100 mM KCl at 95 °C for 10 minutes and slowly cooling the sample to room temperature over several hours, then stored at 4 °C. Serial dilutions of BLM or BLM mutant proteins were incubated with 40 nM oRC32/FAM-oAV320 (partial duplex, Table 1) or oRC75/FAM-oAV320 (G4-dsDNA, Table 1) in helicase assay buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mg/mL BSA, 5 mM MgCl₂, 5 mM ATP, 4 µM oAV322) for 15 minutes at 37 °C in 25 µL reactions. Folded DNA control was obtained by incubating reaction mixture without BLM, and melted control was obtained by omitting BLM and heating reaction to 95 °C for 10 minutes. Five µL of stop buffer (2% SDS, 5 µg/mL proteinase K, 20% (v/v) glycerol, 0.1 mM EDTA) was added to each reaction and 5 µL of each sample was loaded onto a 15% acrylamide 1.5-mm gel in TBE buffer supplemented with 100 mM KCl. Gels were run at 75 V for 1 hour at 4 °C in 1x TBE running buffer with 100 mM KCl. Gels were imaged on an Azure c600 (Azure Biosystems). BLM dsDNA, BLM P868L dsDNA and G4-dsDNA, and BLM K869A K870A dsDNA and G4-dsDNA were done in triplicate. BLM G4-dsDNA, BLM G1120R dsDNA and G4-dsDNA were done in quadruplicate.

Nuclease test: 1 µM BLM or BLM variant was incubated for 30 minutes at room temperature with 40 nM fluorescein labeled dT₃₀ in 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 mg/mL Bovine Serum Albumin (BSA), and 5 mM MgCl₂. Five µL of stop buffer (2% SDS, 5 µg/mL proteinase K, 20% (v/v) glycerol, 0.1 ethylenediaminetetraacetic acid (EDTA)) was added to each sample, and 5 µL of each sample was loaded onto a 15% acrylamide 1.5-mm gel in Tris-Borate-EDTA (TBE) buffer supplemented with 100 mM KCl. Gels were run at 75 V for 1 hour at 4 °C in 1xTBE running buffer with 100 mM KCl and imaged on an Azure c600 (Azure Biosystems).

The saved files are either TIF or CSV files.