Mono-nucleosome Investigation By Single-molecule Magnetic Tweezers

Magnetic tweezers is one of the single-molecule force spectrum techniques,which has the advantages of high throughput and little damage to experimental samples.It can unwrap the internal structure of the sample by exerting force on a single molecule,and investigate the stability mechanism and possible internal structure of the sample at the level of single molecule.In this paper,we mainly use magnetic tweezers to study mono-nucleosome.The main achievements of this paper are as follows.(1)We found that ubiquitination of histone H2AK119 enhances nucleosome stability.Each nucleosome has two H2A histones.When the two H2A histones are ubiquitinated,the rupture force of outer wrap of nucleosome increases from 5pN to 20pN,corresponding to the extension of 20nm.When only one H2A histone was ubiquitinated,the rupture force of outer wrap of nucleosome was unwrapped in two steps: 5pN corresponding to the extension of 10nm and 19pN corresponding to the extension of another 10nm.In other words,ubiquitination of histone H2AK119 can enhance the stability of nucleosome,and mono-ubiquitination can enhance the stability of half outer wrap of nucleosome.Biochemical experiments revealed that ubiquitin did not interact with histone octamer or nucleosome DNA.After crosslinking histone octamer,the experiment of magnetic tweezers showed that the rupture force of inner and outer wrap was consistent with that of non-crosslinked samples,that is to say,ubiquitination did not enhance the stability of histone octamers.Therefore,we speculate that ubiquitin can block the entrance and exit of nucleosomes through the physical steric hindrance in space,so as to realize the function of stabilizing nucleosomes.(2)We found that Swc2,a subunit of SWR1,can selectively recognize H2A nucleosome by single molecule magnetic tweezers.The rupture force of H2A nucleosome of outer wrap was 5pN,and that of inner wrap was 23pN.When Swc2 was added into the system,the rupture force of H2A nucleosome of outer wrap was 2pN and the inner wrap was 5pN.Therefore,Swc2 reduced the stability of the nucleosome.However,Swc2 had no effect on H2A.Z nucleosome.After Swc2 was added,the rupture forces of H2A.Z nucleosome of outer wrap and inner wrap remained unchanged,with the outer wrap 16pN and the inner wrap 25pN.This means that Swc2 is nucleosome selectively.In the process of replacing H2A nucleosome with H2A.Z nucleosome,two domains of H2A histone play an important role,one is M4 and the other is M6.We replace the two domains in H2A histone and H2A.Z histone and find that M4 domain is the main domain in the selective activation of Swc2.On the other hand,we also found that M4 domain plays a major role because it can decide the stability of nucleosome.Because the M4 domain of H2A.Z can enhance the stability of nucleosome,Swc2 is not easy to act on H2A.Z nucleosome.