Biophysical interaction of Bloom syndrome helicase BLM and MCM6 subunit of minichromosome maintenance complex

Abstract

The DNA repair process ensures that DNA damage during DNA replication is minimized so that DNA replication can occur correctly. It is known from previous studies that the interaction between Bloom syndrome protein (BLM) and minichromosome maintenance protein complex (MCM) is important for homologous recombination (HR), one of the DNA repair processes. BLM, one of the RecQ DNA helicase family, plays an important role in the DNA repair mechanism by forming a replication fork complex with other proteins. MCM is also a helicase responsible for unwinding double-stranded DNA during DNA replication. The interaction between the winged helix domain (WHD) of MCM6 and BLM was found in a previous study. The biophysical relationship between BLM and MCM6 is necessary to maintain the normal replication speed after DNA damage. The BLM/MCM6 interaction is a primary factor in the replication stress response and replication repair by DNA double-strand breaks. To perform a series of binding studies in vitro, MCM6 WHD C721S, BLM (220-285), and BLM (220-300) were expressed and purified. We also prepared two peptides (BLM230-238, BLM291-300) of BLM MCM6 binding domain (MBD). The binding affinities between MCM6 WHD C721S and BLM (220-285) and BLM (220-300) were confirmed through ITC experiments. In addition, through the FP experiment, the binding affinity between MCM6 WHD C721S and BLM230-238 (MBD-C) and BLM291-300 (MBD-D) peptides were estimated. To investigate the binding site of MCM6 WHD C721S-BLM interaction in detail, we analyzed the chemical shift perturbations (CSP) of MCM6 WHD C721S during titration with BLM291-300 peptide by using NMR spectroscopy. We confirmed that BLM291-300 is a significant part of the binding site between BLM and MCM6 WHD C721S. In addition, we observed that the hydrophobic interaction is important for binding the two proteins. Based on these results, we suggested detailed biophysical information at the molecular level about the interaction between BLM and MCM6.