Elucidating Recombination Mediator Function Using Biophysical Tools

SIMPLE SUMMARY: This review recapitulates the initial knowledge acquired with genetics and biochemical experiments on Recombination mediator proteins in different domains of life. We further address how recent in vivo and in vitro biophysical tools were critical to deepen the understanding of RMPs molecular mechanisms in DNA and replication repair, and unveiled unexpected features. For instance, in bacteria, genetic and biochemical studies suggest a close proximity and coordination of action of the RecF, RecR and RecO proteins in order to ensure their RMP function, which is to overcome the single-strand binding protein (SSB) and facilitate the loading of the recombinase RecA onto ssDNA. In contrary to this expectation, using single-molecule fluorescent imaging in living cells, we showed recently that RecO and RecF do not colocalize and moreover harbor different spatiotemporal behavior relative to the replication machinery, suggesting distinct functions. Finally, we address how new biophysics tools could be used to answer outstanding questions about RMP function. ABSTRACT: The recombination mediator proteins (RMPs) are ubiquitous and play a crucial role in genome stability. RMPs facilitate the loading of recombinases like RecA onto single-stranded (ss) DNA coated by single-strand binding proteins like SSB. Despite sharing a common function, RMPs are the products of a convergent evolution and differ in (1) structure, (2) interaction partners and (3) molecular mechanisms. The RMP function is usually realized by a single protein in bacteriophages and eukaryotes, respectively UvsY or Orf, and RAD52 or BRCA2, while in bacteria three proteins RecF, RecO and RecR act cooperatively to displace SSB and load RecA onto a ssDNA region. Proteins working alongside to the RMPs in homologous recombination and DNA repair notably belongs to the RAD52 epistasis group in eukaryote and the RecF epistasis group in bacteria. Although RMPs have been studied for several decades, molecular mechanisms at the single-cell level are still not fully understood. Here, we summarize the current knowledge acquired on RMPs and review the crucial role of biophysical tools to investigate molecular mechanisms at the single-cell level in the physiological context.