Study of Interactions of DNA with RECA and Other Proteins.

RecA;Atomic Force Microscopy;Supercoiling;Protein and DNA Interactions;Engineering;Science;Chemical Engineering

Shi, WeixianMayer, Michael ;

University of Michigan

关键词: RecA;    Atomic Force Microscopy;    Supercoiling;    Protein and DNA Interactions;    Engineering;    Science;    Chemical Engineering;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/58526/wshi_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

This thesis describes different methods of stretching DNA using flow fields and investigates the aggregation behavior of RecA-DNA filaments in addition to the preliminary work on the kinetics of RecA-mediated strand exchanges.Using fluorescence microscopy, we compared DNA stretching by droplet evaporation, suctioning of a droplet with pipette tips, and blowing of a droplet with nitrogen or air and found that the best stretching was achieved in fast flows using suctioning and blowing. A statistical analysis of the average length and the surface deposition density of stretched lambda phage DNA molecules showed that both the hydrophobicity of the surface and the pH of the solution affect the stretching of DNA molecules greatly, but the hydrophobic interaction itself is not enough to explain the broad distribution of the stretch observed. The stretched DNA molecules can be used in the study of DNA/protein interactions, as exemplified by our observation of the motion of DNAse I during its interaction with stretched DNA molecules using a dual-color imaging system. We characterized the interwound or so-called ;;supercoiled” aggregates of RecA-DNA filaments using atomic force microscopy. Both RecA-dsDNA and RecA-ssDNA filaments showed mostly left-handedness and we found that single stranded DNA binding protein is necessary for the formation of ordered bundles of RecA-ssDNA filaments. We have suggested that the additional torsional stress generated when filaments approach each other is responsible for this observed coiling of RecA-DNA filaments.We reported the dual roles of ATP hydrolysis and RecA dissociation during strand exchange interactions based on the preliminary experimental observations of the two optimum conditions for fast strand exchanges. While the reaction with a length of 3.7kbp of incoming dsDNA yields maximum products with other conditions maintained, the reaction at 10mM phosphocreatine also produces most products compared with other concentrations. The observed optimums were correlated to the ATP hydrolysis and RecA dissociation during strand exchange interactions. The possible dual roles of these two steps could explain the observation of the two optimums.