RAD52: Paradigm of Synthetic Lethality and New Developments

Abstract

DNA double-strand breaks and inter-strand cross-links are the most harmful types of DNA damage that cause genomic instability that lead to cancer development. The highest fidelity pathway for repairing damaged double-stranded DNA is termed Homologous recombination (HR). Rad52 is one of the key HR proteins in eukaryotes. Although it is critical for most DNA repair and recombination events in yeast, knockouts of mammalian RAD52 lack any discernable phenotypes. As a consequence, mammalian RAD52 has been long overlooked. That is changing now, as recent work has shown RAD52 to be critical for backup DNA repair pathways in HR-deficient cancer cells. Novel findings have shed light on RAD52's biochemical activities. RAD52 promotes DNA pairing (D-loop formation), single-strand DNA and DNA:RNA annealing, and inverse strand exchange. These activities contribute to its multiple roles in DNA damage repair including HR, single-strand annealing, break-induced replication, and RNA-mediated repair of DNA. The contributions of RAD52 that are essential to the viability of HR-deficient cancer cells are currently under investigation. These new findings make RAD52 an attractive target for the development of anti-cancer therapies against BRCA-deficient cancers.

Keywords: Rad52; break induced replication; homologous recombination; single strand annealing; synthetic lethality.

FIGURE 1

Comparison of RAD51 and RAD52-mediated DNA repair pathways. (A) During homologous recombination (HR), the ends of the double strand break (DSB) are resected by nucleases (ex. MRN complex) [see (Zhao et al., 2020) for mechanism of action], exposing single-strand DNA (ssDNA) that becomes bound by RPA. Then the mediator protein, BRCA2 initiates loading of RAD51 on ssDNA helping to displace RPA. RAD51 oligomerizes, forming a nucleoprotein filament, and then searches for the homologous DNA sequence on the intact chromosome. The RAD51 filament invades the intact dsDNA to form a D-loop structure. Further processing by DNA polymerases, chromatin remodelers (ex. RAD54), nucleases, and ligases restore the intact DNA sequence through error-free repair. (B) Alternative to HR, single strand annealing (SSA) begins after resection with the binding of RAD52 to ssDNA. RAD52 promotes the annealing of exposed homologous ssDNA regions on either side of the DSB. Processing of the annealed DNA by nucleases (ex. ERCC1/XPF) results in error-prone repair as the sequences between homologous regions are lost. (C) RAD52 also recognizes and repairs stalled replication forks via break-induced replication (BIR). The structure is cleaved by the endonuclease complex MUS81 and processed by EEPD1 (Kim et al., 2017; Sharma et al., 2020). Bound to the one-ended DNA break, RAD52 invades the dsDNA to form a D-loop. The DNA polymerase contains a non-enzymatic subunit, POLD3, that appears to be specific to this type of repair.

FIGURE 2

Repair of stalled replication forks via BIR. (A) Multiple DNA repair pathways compete to repair stalled replication forks during S/G₂ phase of the cell cycle. (B) Once the cell enters M-phase, unrepaired forks become bound by the FANCD2/FANCI complex. It will attempt to repair the lesion again by a RAD52-dependent BIR-like pathway termed mitotic DNA synthesis (MiDAS). (C) If still unsuccessful, the cell with complete mitosis with each daughter cell inheriting under-replicated ssDNA that is protected by the 53BP1 protein during G₁. (D) In the subsequent S-phase, the cell has one final attempt to repair under-replicated DNA via BIR. After this point, the cells must undergo apoptosis or pass on an incomplete genome.

FIGURE 3

Proposed Mechanisms of RNA-Dependent DSB Repair. (A) Repair of DSBs via inverse RNA strand exchange. Rad52 forms a complex with DSB ends either blunt ended or minimally processed by exonucleases/helicases. Then, RAD52 promotes inverse RNA strand exchange with a homologous RNA transcript. The RNA transcript in the resultant DNA:RNA hybrid provides a template for DNA repair synthesis. The single-stranded tails are removed by flap nucleases, the gaps are filled in, and any remaining nicks are sealed by DNA ligases, restoring the original DNA sequence in an error-free manner. (B) Restart of DNA synthesis stalled at DNA damaged site primed by an R-loop. (C) A tentative role of RAD52 annealing activity in DSB repair. RAD52 promotes annealing between the ssDNA ends of an exonucleolytically processed DSB and homologous RNA transcript. The RNA transcript provides a template for DNA repair synthesis that extends the ssDNA end ensuring an overlap with the ssDNA of another DSB end. This is followed by re-joining of the DSB ends via ssDNA annealing, removal of DNA:RNA heteroduplex by RNase H, filling the gaps by DNA polymerases and sealing the nicks by DNA ligases.

FIGURE 4

Targeting cancer cells via synthetic lethality. PARP inhibitors trap PARP on DNA lesions and suppress repair of ssDNA breaks. This leads to generation of DSBs and other lesions that can only be repaired by HR. Normal cells are capable of repairing these lesions. Dysfunction of BRCA1/2 and related genes cause synthetic lethality with PARP inhibitors so that most of these cells die. Selective pressure forces the cancer cells to become more dependent on alternative RAD52-dependent DNA repair pathways. A combinational treatment of PARP and RAD52 inhibitors enhances the efficacy of each individual treatment via dual synthetic lethality and may cause a delay in the development of cancer drug resistance.