The Research Of Strand Exchange Reaction In Homologous Recombination Process Using Single Molecule Techniques And Monte Carlo Method

DNA is the most crucial genetic material in almost all kinds of organism.In the process of cell division,DNA sequences will be replicated and divided into two cells.The proteins could be generated by processes of transcription and translation,maintaining the normal living processes of cells.However,many kinds of external and internal factors could result into the damage of double strand DNA.To repair the damage of double strand DNA,the cells need to go through the homologous recombination process.RecA protein and Rad51 protein are two kinds of important proteins in the homologous recombination process of prokaryotes and eukaryotes respectively.In this work,we mainly studied how those two proteins work in this process.Homologous recombination process has been studied in detail,mainly including nucleoprotein filament formation,strand search,strand invasion,and strand exchange processes.But the mechanism of strand search and strand exchange is still unclear.In particular,the strand exchange process is difficult to be observed by ordinary experimental techniques for its fast reaction speed and small scale.Therefore,we used single molecule methods to study the strand exchange process.Part homologous and part non-homologous DNA strands were used and defined the position between homologous sequences and non-homologous sequences as a Match Mismatch Joint(MMJ).Magnetic tweezers were used to observe the reaction of RecA near the MMJ which found that most reaction stopped 9 bp before the MMJ and there were lots of reaction steps of 9 bp.So,we hypothesized that 9 bp is the most possible step length of reaction,meanwhile a traveling-wave-like model was established which contains two 9-bp intermediate states.Different step sizes of the strand exchange were counted to get the probability information of the step sizes of the reaction.The reaction of the interface was introduced in the simulation process,and the result of the strand exchange reaction was simulated using the Monte Carlo method.The same MT experiments were also applied to the research of strand exchange reaction of Rad51 protein which had different results compared to RecA protein.To further prove our experiment result under no force conditions,we also designed a nuclease protection experiment.This experiment showed the same result.The F?rster resonance energy transfer(FRET)technology was also used in this study to measure the distance between the marked position of intermediate state.By designing 50%matched sequences,the reaction rate is slowed down to capture the intermediate state of the reaction.Combining the experiment with crystal structure and simulation results,a possible structure of the reaction intermediate state was established.