Recombination Mediators Proteins: History
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Subjects: Physics, Applied
Contributor:
Camille Henry
,
Sarah Henrikus

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.

  • recombination mediator protein
  • single-strand binding protein

1. Introduction

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 belong to the RAD52 epistasis group in eukaryote and the RecF epistasis group in bacteria.

2. Discovery and Phenotype of Bacterial RMPs—RecFOR

In bacteria, RMPs are encoded by recF, recO, and recR genes which belong to the same DNA repair pathway (Table 1). Their discovery is the product of different studies. Horii and Clark were the first to use an Escherichia coli deletion mutant, encoding RecB, RecC, SbcB, and SbcC nucleases, thus allowed them to identify additional genes involved in homologous recombination (HR), including recF [1]. This genetic approach paved the way to identify other members of this pathway, eventually leading to the discovery of recO and recR [2][3]. Due to their phenotypic resemblances, recF, recO, and recR were grouped with other genes (recJ, recQ, recN, …) in the recF epistatic group. Genetic experiments revealing the similarity between SSB overexpression and recF deletion were critical to understand the role of the RecF pathway in SSB removal [4][5]. recFOR deletion mutants are generally characterized by functional defects related to RecA presynaptic nucleofilament (called RecA*) activities, such as: (1) conjugation defect, (2) UV, Gamma rays and drug sensitivity, (3) delayed SOS induction, (4) translesion synthesis (TLS) defects and (5) replication fork instability [1][2][3][6][7][8][9][10][11]. Altogether, these data support a mechanism in which the three bacterial RMPs (RecF, RecO, and RecR) work in the same homologous recombination repair pathway [12][13], mediated by ssDNA (also called gap repair). Interestingly, in bacteria such as Deinococcus radiodurans naturally lacking the recBC genes, the RecF pathway plays a significant role in double-strand break (DSB) repair through an extended synthesis-dependent strand annealing process [14].

Table 1. Overview of recombination mediators and partners in different organisms. This table lists RMPs, their interaction partners and functions in different organisms.

  1. Bacteria 2. Phages T4 or Lambda 3. Eukaryotes
Single-strand binding protein SSB T4 gp32 E.coli SSB RPA
RMP(s) RecF, RecO, RecR UvsY λOrf (NinB) RAD52 BRCA2
Recombinase RecA UvsX λBeta or E.coli RecA RAD51/DMC1 Rad51/DMC1
Pathways facilitated by RMPs HR, SOS, TLS, replication HR and RDR HR, SOS, TLS, replication, phage cycle HR, SSDA HR
Additional partners RecQ, RecJ, RecN, RecG, RecX UvsW helicase λExo and
λMu		 RAD50, MRE11,
XRS2,
RAD54, RAD55,
RAD57, RAD59,
TID1		 RAD50, MRE11,
XRS2, PALB2,
DSS1,
BRCA1

Phylogenetic analysis reveals considerable variability in recF, recO, and recR gene conservation despite their apparent epistasis [15]. In particular, recO or a recO ortholog gene is present in most bacterial species, whereas the recF gene is much less widely distributed [15][16]. This suggests either a redundancy of other DNA repair genes or a more complex mechanism in which RMPs have distinct functions but often cooperate with each other. Consistent with the second possibility, a rising number of studies revealed phenotypic differences between recO and recF in Bacillus subtilis and E. coli [17][18][19][20][21].

3. Discovery and Phenotype of the Bacteriophages RMPs—UvsY/Orf

In bacteriophage T4 (lytic), identification of DNA recombination genes was carried out using genetic screening of deletion mutants presenting sensitivity to UV, DNA damaging agents, such as ethyl and methyl methanesulfonate (EMS and MMS), hydroxyurea (HU), and furthermore showed HR defects [22][23][24][25][26][27]. First, uvsX was identified, then uvsY, and finally uvsW [23][24][26]. Sedimentation, phage plaque, and burst size analysis of single-deletion mutants revealed defects in DNA compaction [23][26][28]. Despite harboring some phenotypic differences, it was established that uvsX, uvsW, and uvsY genes act in the same non-linear DNA repair pathway [26]. The uvsX gene encodes a RecA-like recombinase [22][23][29], whereas uvsW and uvsY [24][26][27][29] encode a DNA helicase and a RMP, respectively. The SSB-like protein of T4 is called gp32 and plays a role in both DNA replication and repair similar to its bacterial homolog [30]. Genetic studies revealed an intricate link existing between UvsY and DNA replication. In the second stage of infection, HR is used as the main source of replication via the recombination dependent replication (RDR) mechanism involving UvsY [31].

The bacteriophage λ (lysogenic) also encodes its own recombination system called λRed able to repair ss- and dsDNA breaks. The λRed combines an exonuclease (5′ to 3′) Exo, a Gam protein, which prevents the action of the host RecBCD and a single-strand binding protein involved in DNA annealing, called Beta [32]. Additionally, λphage encodes an accessory RMP, called λOrf (or NinB) [12][13], dispensable to the λRed recombination mechanism. Due to its broad adaptation to its host, λOrf can substitute for RecFOR in the process of SSB displacement to facilitate RecA loading [12][13][33].

4. Discovery of the Eukaryotic RMP—RAD52/BRCA2

RAD52 was identified as a radiation-sensitive mutation in Saccharomyces cerevisiae [34]. Additional genetic studies revealed its implication in homologous recombination, working alongside other genes (Table 1), all belonging to the RAD52 epistatic group. Genes of this group encode several proteins organized into two subgroups: (1) members of the MRX complex, RAD50, MRE11, and XRS2 involved in the recognition, resection and protection of the DSB; (2) other proteins involved in HR, notably the recombinase RAD51 and RAD51 nucleofilament regulators (RAD54, RAD55, RAD57, RAD59 and TID1). Interestingly, a RAD52 deletion is 250-fold more sensitive to ionizing radiation compared to RAD51 [35]. This greater recombination defect is due to the loss of the second function of RAD52 in DNA repair, via its role in single-strand DNA annealing [36][37]. Although essential for HR in S. cerevisiae, the RAD52 deletion only presented a subtle HR deficient phenotype in vertebrates, suggesting the existence of another DNA repair mechanism in this phylum. Beyond this, recent work uncovered a new mitosis-specific functions of RAD52 in mammals, showing that RAD52 promotes a break-induced replication-like pathway, dubbed mitotic DNA synthesis, that allows completion of DNA replication during chronic stress [38][39][40]. This function appears to be crucial for maintaining of telomeres via recombination [41][42].

The breast cancer susceptibility gene BRCA2 is another crucial gene for HR in eukaryotes and was first identified in humans [43]. Analysis of truncated BRCA2 mutants in mouse uncovered its interaction with RAD51 [44]. The interaction between BRCA2 and the SSB-like protein RPA was established using Hela cells [45]. Together, these observations classify BRCA2 as a RMP facilitating homologous recombination at double-strand breaks during S and G2 phases of the cell cycle, when sister chromatid is available. In Ustilago maydis and mammalian cells, BRCA2 also acts as RMP during meiosis by facilitating the loading of DMC1 (RecA-like) onto ssDNA [46][47]. RAD52 was later found to be synthetically lethal to BRCA2 in humans despite the minor phenotype initially observed for the single-deletion [48]. RMP conservation from one organism to another is varied. Yeast only encodes RAD52, whereas Caenorhabditis elegans only encodes BRC2, mammalian cells encode both and show a different extent in HR phenotypes for the single BRCA2 and double knockdown BRCA2/RAD52 [48][49].

This entry is adapted from the peer-reviewed paper 10.3390/biology10040288

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