Single Molecule Investigation Of DNA And Chromatin Higher Order Structure

This work was carried out mainly from the following three parts.First,we developed high-performance single molecule magnetic tweezers.Secondly,we analyzed a novel structure of chromatin fiber assembled by Me CP2 by magnetic tweezers,and proposed a structure model based on our experiment results.Thirdly,we investigated the dynamics of multi-G-quadruplex at human telomere,demonstrating the existence of interaction between G-quadruplexes.The main contents of our thesis are listed as follow:(1)We developed high-performance magnetic tweezers.Using high-speed CCD camera and microscope with large field of view,the temporal resolution and number of tracking samples were highly improved.We also designed a new controlling software of magnetic tweezers.The newest particle-tracking algorithm was employed to improve the resolution of data,and a PID feedback system was utilized to reduce thermal drift of the device.The new high-performance magnetic tweezers laid the foundation for the follow-up study.(2)Me CP2-Chromatin,a higher order structure of chromatin fiber promoted by Me CP2,was analyzed.We found that Me CP2-Chromatin is different from any known chromatin structure,and has the following characteristics: First,Me CP2-Chromatin has a high degree of cooperativity and will unfold rapidly and completely around 10 p N.Secondly,the unfolding pattern of Me CP2-Chromatin has obvious regularity,which is always alternately unfolded at 50 nm / 100 nm step size,suggesting that the internal assembly pattern is composed of three or six nucleosomes.Finally,Me CP2-Chromatin has a strong reassembly ability,which can be spontaneously reassembled into a chromatin fiber structure after several complete expansions.By truncating the Nterminal and C-terminal of Me CP2 protein,we found that the formation and stability of Me CP2-Chromatin was not affected,but the structure of Me CP2-Chromatin(N-truncated)was more compact and that of Me CP2-Chromatin(C-truncated)was looser in intern assembly pattern.Based on the above experimental results,we proposed a Me CP2-Chromatin assembly model with 6 nucleosomes as a unit,and attempted to explain the molecular mechanism of its unfolding pattern.According to our model,our collaborators have successfully obtained Cryo-EM image of Me CP2-Chromatin assembled by 6 nucleosomes.In addition,we also investigated the effects of pathogenic Me CP2 mutations that can cause Rett syndrome,such as R106 W and P225 R,on chromatin fiber structure.We found that P225 R significantly enhanced the stability of chromatin fiber structure.The results may provide a new insight on the treatment of diseases such as Rett syndrome.(3)We have studied the interaction between multiple G4 at human telomeres using a double G4 system.Our results showed that long telomeric ss DNA could form a continuous G4 structure.Furthermore,we found that there is a stable interaction between G4 s,which has two effects on the multi-G4 system: It enhances the stability of the double G4 system and significantly increases the dwelling time of G4 in the unfolded and folded states.In addition,by mutation of the linked DNA between G4,the interaction between G4 disappears,which suggests that the interaction between G4 needs the participation of linker DNA,and the multi-G4 system without interaction is very unstable.Finally,we proposed that multi-G4 systems loses the part of entropy in free energy,which is the root of the instability of multi-G4 systems.The interaction between G4 can stabilizing multi-G4 system,but the overall stability will still decrease.Therefore,the maximum number of G4 structures will not be formed at the telomere end.This may provide a site for binding of telomeric proteins.