Using Single Molecule Technique To Study The Structures And Dynamics Of DNA And Nucleosomes

The eukaryotic genome DNA is packaged into chromatin by nucleosomes.The nucleosome is the basic repeating unit of chromatin,which is composed of～146 bp of DNA wrapped around an octameric core of histones.The activities of a cell at all times are associated with the expression of DNA,which means that the nucleosome structure and even the chromatin structure are highly dynamic and regulated.The development of single-molecule techniques in the last three decades provides an effective method to explore this highly dynamic structure.The dynamic structure changes of nucleosome and G-Quadruplex(G4)DNA are studied by single molecular magnetic tweezers and single molecular FRET.The dynamic structure of chromatin is mainly regulated by epigenetics,and the main regulatory factors are composed of DNA modification,histone modification,histone variants and chromatin remodeling factors.In this paper,we mainly investigate the effect of ubiquitination on histone and the macro H2A variant(a variant of canonical histone H2A)on cell at the nucleosomal level.In addition,G4,as a special secondary structure of DNA,is formed in the guanine-rich regions of the telomeres and the promoter of some oncogenes,which can directly or indirectly affect the post-translational modification of DNA and histones,thus regulating the chromatin structure.And these epigenetic changes have been linked to a wide range of diseases,including cancer,neurodegenerative and autoimmune diseases.Due to the special structure of G4 and its important function in vivo,more and more anti-cancer drugs are targeting G4 to achieve the treatment of diseases.We first used single-molecule magnetic tweezers to investigate the effect of ubiquitination modification of histone H2B(ub H2B)on the structure of nucleosome.We found that ub H2B can slightly reduce the stability of nucleosome.However,with the help of FACT(Facilitates Chromatin Transcription),the ub H2B-nucleosome can form an extremely stable structural state that may facilitate transcription.We found that FACT prefers to bind and deposit H2A/ub H2B dimers to form nucleosome.Moreover,the well binding of the FACT on the ub H2B-nucleosome greatly enhances the stability and maintains the integrity of the nucleosome.ub H2B recruits FACT by enhancing the association between FACT and ub H2B-nucleosome,at the same time,promotes transcription elongation by forming a stabilized structural state at nucleosome level with the help of FACT.Then we used single molecule magnetic tweezers to study the effect of macro H2A variant of canonical histone H2A on the nucleosome.We found surprisingly that such a large macro H2A variant had no effect on the mechanical stability and unfolding dynamics of the nucleosome compared to the canonical histone H2A.In the presence of FACT,the mechanical stability of macro H2A-nucleosomes is significantly reduced,and FACT is no longer able to reassemble fully unfolded macro H2A-nucleosome compared to nucleosome formed by canonical histone.Further studies showed that the effect on the reassembly ability of FACT is in the linker region of the macro H2A variant,and we pinpointed this key factor in two amino acid sites,Ser-139 and Gln-140.To our surprise,the removal of the C-terminal tail of canonical histone H2A and its variants H2A.X and macro H2A did not affect the mechanical stability and unfolding dynamics the nucleosome itself.In the presence of FACT,the magnetic tweezers experiment of variant H2A.X simulating phosphorylation in DNA damage repair showed the same results as macro H2A,which provided us new ideas to understand the mechanism of DNA damage repair,and also allowed us to see the cross-talk between histone variants.Finally,we investigated the effect of Curaxin CBL0137 on the structure of G4using single molecular FRET.Experimental results showed that CBL0137 could induce G4 to fold into a parallel structure,and this effect was independent of the concentration of K~+.Further changing the loop length in the G4 sequence reveals that the folding of G4 becomes unstable as the loop becomes longer,and the addition of drug CBL0137 could facilitate and stabilize the folding of G4.This provided clues for us to understand the potential therapeutic mechanism of CBL0137,and also provided a meaningful reference for designing drugs targeting G4.