Single molecule investigations into DNA replication, recombination and repair

Double-stranded DNA breaks (DSBs) is a principal cytotoxic damage that can be repaired by several well-known repair pathways. One of the most prominent routes is the homologous recombination (HR). The mechanism of repair has been studied primarily by biochemical analysis over the years. Recent advances in single molecule techniques has enabled researchers to investigate the repair mechanisms at single protein level with nanometer resolution and millisecond time scale. The ability to fluorescently label different parts of the protein or protein complex allows us to monitor in real-time conformational changes as well as the mechanistic details with which the proteins target DNA damage and conduct repair. Srs2 dismantles presynaptic Rad51 filaments and prevents its re-formation as an anti-recombinase. However, the molecular mechanism by which Srs2 accomplishes these tasks remains unclear. Here we report a single-molecule fluorescence study of the dynamics of Rad51 filament formation and its disruption by Srs2. Rad51 forms filaments on single-stranded DNA by sequential binding of primarily monomers and dimers in a 5’–3’ direction. One Rad51 molecule binds to three nucleotides, and six monomers are required to achieve a stable nucleation cluster. Srs2 exhibits ATP-dependent repetitive motion on single-stranded DNA and this activity prevents re-formation of the Rad51 filament. The same activity of Srs2 cannot prevent RecA filament formation, indicating its specificity for Rad51. Srs2’s DNA-unwinding activity is greatly suppressed when Rad51 filaments form on duplex DNA. Taken together, our results reveal an exquisite and highly specific mechanism by which Srs2 regulates the Rad51 filament formation. Trinucleotide repeat (TNR) expansion is the root cause for many known congenital neurological and muscular disorders in human including Huntington’s disease, Fragile X syndrome and Friedreich’s ataxia. The stable secondary hairpin structures formed by TNR may trigger fork stalling during replication, causing DNA polymerase slippage and TNR expansion. Srs2 and Sgs1 are two helicases in yeast that resolve TNR hairpins to various degrees during DNA replication and prevent genome expansion. Using single molecule fluorescence, we investigated the unwinding mechanisms by which Srs2 and Sgs1 resolve TNR hairpin and compared it to the unwinding of duplex DNA. While Sgs1 unwinds both structures indiscriminately, Srs2 displays a repetitive unfolding of TNR hairpin without fully unwinding it. Such activity of Srs2 shows dependence on the folding strength and the total length of TNR hairpin. Our results reveal disparate molecular mechanism of Srs2 and Sgs1 that may contribute to efficient resolving of the TNR hairpin. We use single molecule fluorescence assays and ensemble measurements to study yeast and human proteins. Single molecule assays include smFRET (single molecule Förster Resonance Energy Transfer) assay which utilizes two fluorescent dyes (Cy3 and Cy5), and smPIFE (single molecule Protein Induced Fluorescence Enhancement) which employs the use of the Cy3 dye only and offer a sensitivity range (0-4 nm) that is finer than what could be achieved with FRET (2-8 nm) (1). The main goals of the project are to investigate the key steps involved in the DNA replication & repair pathways as well as gene regulatory control pathways in order to gain a better understanding of the roles the aforementioned proteins play in maintaining proper cellular functions