Single-Molecule Study On BLM Unfold G-Quadruplex

The single-molecule techniques have made a huge progress in molecular biology research in recent years.Magnetic tweezers,one of the single-molecule manipulation techniques,gained favour in researchers for its simplicity and high resolution.But the resolution of traditional magnetic tweezers is limited by Brown motion of magnetic beads while the force was lower than 10 pN.In order to improve the resolution,we introduced total internal reflection fluorescence techniques into magnetic tweezers,and we developed a novel single molecule connection: “magnetic bead-DNA handle-fluorescent bead-DNA substrates”.The improved magnetic tweezer could realize a nanometer-scale spatial resolution under a small force,and meantime keep a high time resolution in a long time observation.G-quadruplex(G4)is a DNA structure which commonly exists in human genome.It is considered as a significant structure in DNA metabolism such as replication,transcription and homologous recombination.As the single molecule technique was developed,more and more studies of G4 were reported in recent years.But there are still some basic problems unclear.The G-quadruplex helicases were widely researched these years.One of the most thoroughly studied in them was BLM helicase.The BLM helicase functions in homologous recombination repair to prevent genomic instability.BLM is highly active in binding and unfolding G4.Here we used total internal reflection fluorescence high resolution magnetic tweezer with illumination of total internal reflection fluorescence to research the process of BLM resolving G4.We found the process is stepwise.Our apparatus could get a long time results compare to the single molecule fluorescence technique.So we performed experiments with a nearly saturated ATP concentration.We found BLM have two ways to maintain G4 dissolution.We also performed single molecule fluorescence resonance energy transfer experiment in the same condition,we found that the 2-3 pN force in magnetic tweezers have rare influence in the process of BLM interact with G4.We studied the dwell time of BLM stayed in unfolded G4,indicated that the two pathway of BLM's maintenance of G4 dissolution are correspondent to a long dwell time process and a short dwell time process.We found that the ATP hydrolysis and the process of HRDC domain binding in ssDNA were responsible for the long dwell time process.Our data enabled us to proposed a model in which the HRDC domain functions as a regulator of BLM,depending on the position of the HRDC domain of BLM in action: When HRDC binds to the G4 sequence,BLM may hold G4 in the unfolded state;otherwise,it may remain on the unfolded G4 transiently such that G4 can refold immediately.