Elucidating Recombination Mediator Function Using Biophysical Tools

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.

Catabolite control protein C contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes

“…These genes include DNA recombination proteins (recU, recA, recF, recR, and recO), holliday junction ATP-dependent DNA helicase (ruvA, ruvB), lexA repressor, replication and repair (dnaA, dnaD, and dnaE), DNA polymerase (polA and holB), and DNA mismatch repair proteins (mutM and mutS). These genes are involved in various processing steps of DNA replication and play roles in the restarting of stalled replication forks, homologous recombination, the repair of double-stranded DNA breaks, and the introduction of adaptive point mutations (Koroleva et al, 2007;Henry and Henrikus, 2021;Matsuda et al, 2022). These results indicate that the increased expression of DNA damage repair proteins in the absence of CcpC regulation improves the response of ∆ccpC bacteria to oxidative stress-induced DNA damage.…”

“…Previous studies have shown that bacterial responses to DNA damage are mediated by the SOS response pathway, which promotes the repair and survival of DNA-damaged bacteria and the induction of genetic variation in stressed and stationary-phase bacteria (Matsuda et al, 2022). In L. monocytogenes, the SOS response pathway is regulated by LexA and RecA (Henry and Henrikus, 2021), and expression of L. monocytogenes recombination proteins is elevated in response to DNA-damaging agents (Ojha and Patil, 2020). Indeed, studies of stress-related survival have shown that a ΔrecA L. monocytogenes mutant is less resistant to heat, H 2 O 2 , and acid exposure relative to wild-type strain.…”

Insight into Single-Molecule Imaging Techniques for the Study of Prokaryotic Genome Maintenance

“…This enhanced signal-to-noise ratio produces highcontrast images of selectively illuminated fluorophore samples. 23,24 In the context of prokaryotic genome maintenance, TIRF microscopy has been widely employed to visualize DNA replication, 26−30 transcription, 31,32 translation, 32 DNA repair, and recombination, 33 and cell segregation during the bacterial cell cycle in real-time. 34 To study individual molecules using TIRF microscopy, the molecules of interest must be fluorescently labeled and immobilized on a surface.…”

“…This technique has found widespread application in the study of subpathways of DNA repair and recombination processes within bacterial cells. 33 2.2.2. Super-Resolution Microscopy.…”

“…Single-molecule studies have shown that, prior to homologous recombination, various recombination mediator proteins (RMPs) facilitate the loading of RecA onto the DNA, promoting homologous recombination. 33 For a detailed review of how RMPs promote homologous recombination in various subpathways see Henry et al 2021 and Whinn et al 2021. 67,92 One important pathway in facilitating recombination the RecFOR pathway.…”