Study Of The Interactions Between Sso7d And DNA By Single-molecule Techniques

Gene is the basic unit of inheritance.It plays an important role in regulating biological processes,such as DNA transcription,replication,virus infection and immunity.Therefore,to maintain biological processes working properly,gene structure should be stable.Gene is a fragment of DNA,which has heredity function.To maintain the stability of gene,DNA is organized and compacted to smaller and more stable structure by chromatin proteins in all three kingdoms of life(Bacteria,Archaea and Eukarya).For example,DNA is organized to nucleosome by histone in Eukaryotes.Researching the mechanism of interplay between chromatin protein and DNA is important to understand the stability of genetic inheritance.Moreover,it is also helpful to utilize the mechanism to produce synthetic bio-composites.Sulfolobus is a kind of Archaea.Its natural habitat is usually a high temperature environment with sulfur.The most suitable growth environment is 80 centigrade.Generally,DNA structure is hard to be stable in such environment.However,chromatin proteins in Sulfolobus has an important role in maintaining DNA structure stability in that extreme environment.The protein Sso7 d,which is studied in this thesis,is a kind of chromatin protein in Sulfolobus.Previous studies have shown that Sso7 d is important for DNA stability.Binding of Sso7 d constrains negative supercoils of DNA,and increases melting temperature compared with bare DNA.In this thesis,advanced single molecule technologies,such as magnetic tweezers and atomic force microscope,are used to study the interplay between Sso7 d and DNA.Data from atomic force microscope experiments could reveal how Sso7 d and DNA interact.The binding of Sso7 d results in a DNA bend and decrease of DNA persistence length.DNA is compacted through binding with Sso7 d and becomes more compact in high concentration of Sso7 d.Our experiments reveal the process of forming a loop structure in DNA.We also provide the explanation of this process.By magnetic tweezers experiments,we study DNA force–extension curves impacted by binding of Sso7 d.Elastic coefficient of DNA decreases with binding more Sso7 d.Moreover,we also measure the force of unzipping the duplex DNA indifferent concentrations of Sso7 d.Binding of Sso7 d increases the unzipping force of duplex DNA.We calculate reaction rate parameters of the interaction between Sso7 d and DNA,and the difference of binding energy with and without binding of Sso7 d.These results are helpful in understanding the reason of DNA stability in high temperature.In addition,we also study how Sso7 d influences DNA helicase by magnetic tweezers experiment.First,we observe the process of helicase BLM unwinding double stranded DNA in absence of Sso7 d.Then,we add Sso7 d and observe the unwinding process.Our results show that the combination of Sso7 d and DNA can pause the unwinding process.