Rad52 mediates class-switch DNA recombination to IgD

While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig class remains unknown. In B cells, IgD is expressed together with IgM as transmembrane receptor for antigen through alternative splicing of long primary VHDJH-Cμ-s-m-Cδ-s-m RNA, which also underpins the secreted form of IgD. IgD is also expressed through class switch DNA recombination (CSR), as initiated by AID-mediated double-strand DNA breaks (DSBs) in Sμ and σδ and resolution of such DSBs by a yet unknown alternative endjoining (A-EJ) mechanism. This synapses Sμ with σδ region DSB resected ends leading to insertion of extensive S-S junction microhomologies, unlike the Ku70/Ku86-dependent NHEJ which resolves DSB blunt ends in CSR to IgG, IgA and IgE with little or no microhomologies. We previously demonstrated a novel role of DNA annealing homologous recombination Rad52 protein in 'short-range' microhomology-mediated synapsis of intra-Sδ region DSBs. This led us to hypothesize that Rad52 is also involved in the short-range microhomology-mediated A-EJ recombination of Sμ with σδ. We found that induction of IgD CSR by T-dependent or T-independent stimuli downregulated Zfp318 (the suppressor of Cδ-s-m transcription termination), promoted Rad52 phosphorylation, recruitment of Rad52 to Sμ and σδ leading to Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Further, Rad52 knockdown in human B cells virtually abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and lupus patients. Thus, Rad52 mediates CSR to IgD by synapsing Sμ-σδ resected DSB ends through microhomology-mediated A-EJ and in concert with Zfp318 modulation. This is a previously unrecognized, critical and dedicated role of Rad52 in mammalian DNA repair.

DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2–Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Physiological and Pathological Roles of RAD52 at DNA Replication Forks

Understanding basic molecular mechanisms underlying the biology of cancer cells is of outmost importance for identification of novel therapeutic targets and biomarkers for patient stratification and better therapy selection. One of these mechanisms, the response to replication stress, fuels cancer genomic instability. It is also an Achille’s heel of cancer. Thus, identification of pathways used by the cancer cells to respond to replication-stress may assist in the identification of new biomarkers and discovery of new therapeutic targets. Alternative mechanisms that act at perturbed DNA replication forks and involve fork degradation by nucleases emerged as crucial for sensitivity of cancer cells to chemotherapeutics agents inducing replication stress. Despite its important role in homologous recombination and recombinational repair of DNA double strand breaks in lower eukaryotes, RAD52 protein has been considered dispensable in human cells and the full range of its cellular functions remained unclear. Very recently, however, human RAD52 emerged as an important player in multiple aspects of replication fork metabolism under physiological and pathological conditions. In this review, we describe recent advances on RAD52’s key functions at stalled or collapsed DNA replication forks, in particular, the unexpected role of RAD52 as a gatekeeper, which prevents unscheduled processing of DNA. Last, we will discuss how these functions can be exploited using specific inhibitors in targeted therapy or for an informed therapy selection.

RAD52: Viral Friend or Foe?

Mammalian Radiation Sensitive 52 (RAD52) is a gene whose scientific reputation has recently seen a strong resurgence. In the past decade, RAD52, which was thought to be dispensable for most DNA repair and recombination reactions in mammals, has been shown to be important for a bevy of DNA metabolic pathways. One of these processes is termed break-induced replication (BIR), a mechanism that can be used to re-start broken replication forks and to elongate the ends of chromosomes in telomerase-negative cells. Viruses have historically evolved a myriad of mechanisms in which they either conscript cellular factors or, more frequently, inactivate them as a means to enable their own replication and survival. Recent data suggests that Adeno-Associated Virus (AAV) may replicate its DNA in a BIR-like fashion and/or utilize RAD52 to facilitate viral transduction and, as such, likely conscripts/requires the host RAD52 protein to promote its perpetuation.

DSS1 interacts with and stimulates RAD52 to promote the repair of DSBs

The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as another interacting partner of DSS1 and shows that both proteins are important players in the SSA and BIR pathways of DSB repair.

A Spotlight on Rad52 in Cyanidiophytina (Rhodophyta): A Relic in Algal Heritage

The RADiation sensitive52 (RAD52) protein catalyzes the pairing between two homologous DNA sequences’ double-strand break repair and meiotic recombination, mediating RAD51 loading onto single-stranded DNA ends, and initiating homologous recombination and catalyzing DNA annealing. This article reports the characterization of RAD52 homologs in the thermo-acidophilic Cyanidiophyceae whose genomes have undergone extensive sequencing. Database mining, phylogenetic inference, prediction of protein structure and evaluation of gene expression were performed in order to determine the functionality of the RAD52 protein in Cyanidiophyceae. Its current function in Cyanidiophytina could be related to stress damage response for thriving in hot and acidic environments as well as to the genetic variability of these algae, in which, conversely to extant Rhodophyta, sexual mating was never observed.

Structure of the human DNA-repair protein RAD52 containing surface mutations

The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD521–212) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD521–212containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD521–212was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD521–212rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD521–212ring and stabilized the ring–ring association observed in the crystal.