INVESTIGATION OF REGULATORY MECHANISM OF DNA HOMOLOGOUS RECOMBINATION REPAIR AT SINGLE MOLECULAR LEVEL

Abstract

Homologous recombination (HR) repairs DNA double strand break through genetic exchange with identical chromosome under the regulation of a variety of repair proteins. Single-stranded DNA binding proteins such as prokaryotic SSB and its eukaryotic homolog RPA are the most prevalent proteins that have important roles in HR. In this thesis, regulatory role of force on the properties and dynamics of E.coli SSB and human RPA on ssDNA binding is investigated. In addition, a label-free single molecule platform is developed to detect annealing events between mechanically extended ssDNA and short complementary ssDNA oligos in real time. SSB and RPA showed different extent of inhibitory effect on annealing between ssDNA through retarding the nucleation process. Moreover, a parallel triple-stranded DNA complex is observed to form between mechanically extended ssDNA and homologous dsDNA, which has similar helical rise and stability compared to B-form dsDNA. This triple-stranded complex provides insights for the mechanism of homology recognition and pairing during HR.