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Effects Of Psoralen On Histone-DNA Interactions Studied By Using Atomic Force Microscopy

Posted on:2021-02-19Degree:MasterType:Thesis
Country:ChinaCandidate:Y Q ZhangFull Text:PDF
GTID:2404330623978398Subject:Polymer Chemistry and Physics
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The investigation of the DNA-histone interactions and factors that affect such interactions in the nucleosome is essential for understanding the role of chromatin organization in all cellular processes involved in the repair,transcription,and replication of the eukaryotic genome.As a kind of photosensitive matter,psoralen is used in the treatment of skin disease with ultraviolet light(PUVA).The effect of treatment is remarkable,but the side-effect is also obvious.Psoralen can be embedded in 5' TA sequence in double-stranded DNA(dsDNA),and dsDNA is mainly wrapped around histone octamer to form nucleosome structure in human cells.Therefore,it is very necessary to explore the influence of psoralen on DNA-histone interactions.Atomic force microscopy(AFM)imaging is one of the efficient methods for the study of biomacromolecules and their interactions with their binding molecules at single molecule level.In this thesis,we use atomic force microscopy(AFM)imaging to visually investigate the effects of psoralen on histone-DNA interactions,and further explore the effect of salt concentration on the histone-DNA complexes.In chapter 1,the nucleosome was reviewed briefly.The histone octamer(two of each H2 A,H2B,H3 and H4)is wrapped with 147-base-pair(bp)of DNA to form a nucleosome.Because 75-90% of genomic DNA wraps around histones to form nucleosomes,which can eventually form very compact chromosome via multi-scale assembly,as a result,a single human cell can accommodate ~2 meter-long-DNA.Then,psoralen and its interaction with dsDNA was introduced.Psoralen is a kind of photosensitive matter that can selectively intercalate and crosslink the 5'-TA dinucleotides of double-stranded DNA in the presence of long wave length ultraviolet light(UVA),which has been widely used for the treatment of skin disease(e.g.psoriasis)with UVA.In the last part of this chapter,the working principle,instrument composition,and working mode of the AFM are briefly introduced.And the application of AFM imaging technology in the field of biological macromolecule research is briefly summarized.In chapter 2,we used the EM-PCR method to prepare the long DNA fragments containing(TATAAACGCC)n repeating sequences.The repeat sequences were ligated with the T-Vector and corresponding plasmid DNAs were screened by standard molecular biology method.The obtained recombinant plasmid DNA was then linearized by different kinds of enzymes,leaving the specific histone-binding sequence at different locations of the dsDNA.The above prepared three kinds of histone-binding DNA were mixed with equal molar amounts of histone and assembled into nucleosomes by using the salt dialysis method.To compare the ability of histone binding with specific and random sequences,we counted the location of nucleosome formed in different DNA substrate.AFM imaging show that histone proteins preferentially bind to the target histone-binding sequences,since the locations of the complexes correlate well with the specific substrate.These results demonstrate that the inserted repeat sequence can bind to histone proteins more easily in vitro.In order to check the effect of psoralen on histone-DNA interactions,we selected BsaI-digested recombinant plasmid(with the histone-binding sequence locate at the middle of the DNA chain)as the substrate and performed the assembly in the absence and presence of psoralen with or without UV crosslinking treatment.The complexes were characterized by AFM imaging.Our results revealed that the presence of psoralen increased the probability of interactions between histone proteins and random nonspecific DNA sequences.Upon the introduction of UVA irradiation,the chance of nonspecific-sequence binding is further increased.The experiment results showed that the presence of psoralen altered the binding modes of histone-DNA and caused the formation of histone-DNA complexes on random DNA sequences.In chapter 3,in order to compare the stability of nucleosome structure formed by both specific and random sequences.The above three kinds of nucleosomes were dialyzed in the solution of high ionic strength(named as reverse dialysis).After the reverse dialysis,the three samples were characterized by AFM imaging.Our results show that after the treatment by high salt,the nucleosome formed by the random sequences were preferentially disassembled.Statistical analysis results show that for bare DNA(no psoralen)high ionic strength has no effect on the ratios of specific-sequence binding,while in the presence of psoralen the high ionic strength can greatly reduce the probability of nonspecific-sequence binding.Further addition of the UVA light,the probability of nonspecific-sequence binding is further reduced.This phenomenon indicates that the nucleosome formed by the random sequences are not as stable as that assembled by specific sequences.To explore the stability and integrity of the structure formed by histone and different types of DNA sequences,we compared the volume of the complexes and the angles of the DNA arms of the complexes formed by the specific sequences and the common sequences.The calculated results show that the volumes of the complexes formed by histone and specific sequence are obviously larger than random sequence.The much smaller angle formed by the specific sequence means that the specific sequences wrapped tightly around histone octamer,while the random sequences form loose and incomplete wrapping around the histone octamer.These two facts together indicate that the specific DNA sequence binds more efficiently with histone protein and forms more complete nucleosome structure than nonspecific random sequences.Our results show that we can tune the assembly and disassembly process of nucleosome in vitro by DNA sequence,psoralen and salt concentration.In addition,our results indicate that during the PUVA treatment the presence of psoralen may facilitate the formation/stabilization of nucleosome and thus affect corresponding biological processes,such as the DNA replication gene expression.Our findings may provide an alternative explanation on the side-effect of psoralen drugs in the treatment of diseases.
Keywords/Search Tags:psoralen, histone-DNA interactions, nucleosome, atomic force microscopy
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