Single-Molecule Insight Into Dynamics Of DNA Replication And Chromatin Remodeling

Deoxyribonucleic acid(DNA),as the carrier of genetic information,is an important macromolecule in cells and its replication can affect cell growth and division.DNA replication in cells is mainly accomplished by DNA polymerases,which attain high fidelity to maintain genetic stability.Exonuclease proofreading activity can significantly improve polymerase fidelity;on the other hand,excessive excision can also affect the efficiency of polymerization.Therefore the investigation of exonuclease activity of polymerases is very important in the biophysical field.In eukaryotic cells,DNA is wrapped around histones to form nucleosomes,which must slide out from histones to be replicated,and chromatin remodeling factors are the motor proteins that accomplish this process.The investigation of the mechanisms of DNA translocation by remodeling factors is a hot topic in epigenetic research.Single-molecule fluorescence resonance energy transfer(sm FRET)is a real-time observation method with high resolution can measure the dynamic process of DNA replication and chromatin remodeling.In this thesis,we used this method to study the above two topics.When studying the dynamics of DNA replication,we selected T7 bacteriophage DNA polymerase gp5 and Escherichia coli DNA polymerase I Klenow fragment(KF)for investigation.gp5 opens the double-strand DNA to attain the 3'—5'strand(leading strand)and continuously copy this template.Another pattern is called extension synthesis: DNA polymerase copy the separated 5'—3' strand(lagging strand)in a discontinuous manner.It's found that gp5,without the help of external force,repeat in the synthesis-excision cycle which leading to less product of strand-displacement synthesis.It's established that DNA annealing force can regulate the length of strand-displacement synthesis.Further experiment indicated that T7 DNA helicase gp4 reduce the annealing force which enable gp5 to have processive strand-displacement synthesis.KF,as a polymerase with high fidelity and exonuclease activity,mainly synthesized and repaired DNA with strand-displacement synthesis.To investigate its exonuclease activity,we used the abasic lesion(Ap)to increase the probability of exonuclease activity.Ap has been found to affect polymerase and exonuclease activity of KF at multiple base-pair sites.By analysis of dynamic time,we revealed that the transfer from polymerase activity to exonuclease activity is imitated from pre-translocation state and the transfer from exonuclease activity to polymerase activity would induce KF to backslide 1 bp.This dynamic of transfer enhance the fidelity of KF and remain the efficacy of polymerase activity of KF.While investigating the mechanisms of chromatin remodeling,we selected Snf2 and Chd1 chromatin remodeling factors from yeast cells.Due to evolutionary conservation,mechanisms of these 2 factors are close in higher animals and yeast,and investigating these mechanisms could also help human better understand the physiological metabolism and disease.Snf2 translocate DNA out of the nucleosome which leading to enable DNA replication.In this thesis,by using sm FRET,we found that the mechanism of DNA translocation by Snf2 is as follows: Firstly Snf2 binds to nucleosomes and translocate lagging-strand DNA from the entry-side to from a 1 base pair(bp)twist.With ATP bound,Snf2 undergoes a conformational change and deliver the DNA twist to exit-side.Chd1 senses the linker DNA and induces arrangement of nucleosome which play an essential role in chromatin regulation.Here we found the exit-side DNA is unwrapped by Chd1.In the unwrapped state,Chd1 did not induce 1bp twist when ADP was bound,which indicate inhibition of the translocation activity.