Mechanistic studies of the human recombination proteins RAD51 and RAD54 at the molecular level via fluorescence resonance energy transfer (FRET).

摘要

Homologous recombination (HR) is a key double stranded DNA break repair process responsible for maintaining genetic stability and creation of diversity. During Meiosis I, HR mediates rearrangement in sister chromosomes, allowing for the creation of genetic variability in most organisms. Also, during Mitosis, HR plays a very important role in maintaining genomic stability and avoiding lethal mutations through accurate repair of certain DNA damages. The human Rad54 (hRad54) and human Rad51 (hRad51) proteins are key proteins required for proper HR. hRad51 forms a right-handed single stranded DNA (ssDNA) filament and promotes homologous pairing and strand exchange in an ATP dependent manner. hRad54 belongs to the Swi2/Snf2 family of DNA dependent ATPases and amongst other activities, it has chromatin remodeling activity and it has been found to promote Holliday junction branch migration. hRad51 and hRad54 are known to directly interact and enhance each other's known activities.;This dissertation focuses on studying the strand exchange and branch migration steps of the HR mediated DNA repair pathway. We have developed Fluorescence Resonance Energy Transfer (FRET)-DNA assays in order to sensitively monitor strand exchange and branch migration reactions in real time. In one set of experiments we have focused on attempting to establish the specific roles hRad51 and hRad54 play during the HR mediated double strand break repair (DSBR) pathway at the naked DNA level. Generally, DNA in the cell is wrapped around core histones resulting in a structure called the nucleosome. Here we also created a DNA-FRET system that allows real time strand exchange studies to be performed at the nucleosome level.;Our results indicate that hRad54 greatly stimulates the overall efficiency of hRad51 mediated strand exchange probably by stabilizing the hRad51-ssDNA filament, the active form of Rad51. We also find that hRad54 can promote branch migration of a Holliday Junction (HJ) through various degrees of mismatches more efficiently than the same reactions promoted by hRad51. The results imply that the main role of hRad51 in HR is in the homology recognition and initial strand exchange reaction to form a three-stranded Holliday junction. After the formation of a three-stranded Holliday junction which subsequently becomes a four-stranded Holliday junction, hRad54, which promotes more efficient branch migration, could take over to promote long range strand exchange. Our results from our FRET-nucleosome system show that hRad51 and hRad54 mediate strand exchange efficiently when the donor double stranded DNA (dsDNA) substrate is present as naked DNA. The presence of the same donor dsDNA molecule as a core nucleosome prevents hRad51 and hRad54 mediated strand exchange. Our data provides evidence that hRad51 and hRad54 are not sufficient to mediate strand exchange when the donor molecule is present as a core nucleosome suggesting the possible involvement of other factors during strand exchange in vivo.;Lastly, in this dissertation we have taken advantage of the powerful atomic force microscopy (AFM) technique to visualize interactions involving the HR proteins RPA, and Brh2 and a specially designed DNA substrate. Previous results have indicated, through a set of experiments using radioactively labeled DNA and gel electrophoresis, the binding of RPA and Brh2 to specific regions on a partially double stranded and partially single stranded circular DNA substrate (Gapped DNA). This substrate represents a key intermediate formed during HR mediated DNA repair. Here we show supporting evidence by visually demonstrating the specific binding of RPA to the single strand DNA region on the Gapped DNA substrate and the specific binding of Brh2 to the single strand-double strand junction site on the Gapped DNA substrate by means of AFM.