Study of Bloom resolving G-quadruplex process by using high resolution magnetic tweezer with illumination of total internal reflection

Abstract

G-quadruplex (G4) is a DNA structure which commonly exists in human genome, and it is considered as an important structure in DNA metabolism such as replication, transcription and homologous recombination. The G-quadruplex helicases have been widely investigated these years. Of them, the Bloom (BLM) helicase is most thoroughly studied. However, there are some basic problems that are still unclear. Most of previous studies of G4 are performed by single molecule fluorescence resonance energy transfer technique. The G4 is in a free state in these experiments, which is different from the physiological environment in cells. The traditional magnetic tweezers have a limitation of spatial resolution in a low force circumstance. Thus here we use high resolution magnetic tweezer under the illumination of total internal reflection fluorescence to study the process of BLM resolving G4. Our modification of magnetic tweezer is to separate the measurements of force and distance of magnetic tweezer in order to improve the spatial resolution, which allows us to observe the unfolding process of G4. With a 2-3 pN force we find that the process of BLM unfolding G4 in low ATP concentration is stepwise, and the G4 is mainly in the state between G-quadruplex and G-triplex. We also find that the BLM could interact with G4 for a long time. Our apparatus is also able to obtain the long time observation results compared with the single molecule fluorescence technique. So we perform experiments with a nearly saturated ATP concentration. We find that the BLM has two ways to maintain G4 dissolution in this condition. The BLM could unfold the G4 repetitively in a long period and it could also keep the G4 in unfolding state for a long time after it has opened the G4. Finally, we also perform single molecule fluorescence resonance energy transfer experiment in the same condition, and we find that the 2-3 pN force in magnetic tweezers has a rare influence on the process of BLM interacting with G4. The results of single molecule fluorescence resonance energy transfer experiments are corresponding to the results of magnetic tweezer in the same conditions. All of our experimental results show that ATP dependent BLM has a high affinity with G4 and BLM has a different way to resolve G4 in high ATP concentration. These results could provide new ideas of the mechanism of BLM resolving G4. Our modified magnetic tweezer shows its capacity in G4 single molecule study, and it could be a useful tool in the future single molecule studies.