| Summary: | 碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 107 === RecA protein is essential for repairing the damaged chromosomal DNA by mediating homologous recombination. RecA can bind on ssDNA to form a nucleoprotein filament. Later, a RecA nucleoprotein filament can search for homologous sequence from another duplex dsDNA. The last step is the RecA dissociation after the completion of strand exchange. Previously, Cox et al. reported that a strain expressing C-terminal truncated RecA protein does not exhibit more UV-sensitive than a wild type strain. RecA ΔC17 possesses a higher ssDNA binding affinity than the wt RecA. Moreover, the deletion of residues in the C-terminus of RecA will shift the pH reaction profile of strand exchange to a high pH condition. The C-terminus of most RecA proteins from Deinococcus genus is shorter and lack of negative-charged residues than that of E. coli RecA. D. ficus RecA, which has 92% sequence identity to Dr. RecA, was found to possess a similar ATPase activity and a similar DNA strand exchange reaction pathway to those of E. coli RecA. The pH profile of DNA strand exchange efficiency is shifted toward higher pH for D. ficus RecA. Here, we replaced the last 17 amino acid of D. ficus C-terminus with that of E. coli RecA to construct a chimera protein, D. ficus- C17E. coli RecA. Previously, we found that the rate-limited step is nucleoprotein formation in the strand exchange process for E. coli RecA and D. ficus-C17E. coli RecA with TPM experiment. In contrast, D. ficus RecA and E. coli ΔC17 RecA possess higher DNA binding affinities and lower protonation requirements for the formation of nucleoprotein filament. In order to investigate the influence of C-terminal residues in the formation of nucleoprotein filament, we would like to apply FRET experiment to observe dynamic and stability of RecA nucleoprotein filament in detail.
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