Data for: Biochemical properties of naturally occurring human bloom helicase variants

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 P86..., Differential Scanning Fluorimetry: Differential scanning fluorimetry (DSF) was carried out as previously described with the following modifications. WT BLMcore, BLMcore P868L, or BLMcore 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 Tm by determining the maximum of the first derivative (dRFU) on DSFWorld. ∆Tm val..., The saved files are either TIF or CSV files.