Full-length RAD52 exists as a heptameric ring, while purified NTDs have been shown to form an undecameric ring structure
[41,42][41][42]. While a ring structure would usually be suggestive of a helicase, the DNA-binding domain of RAD52 is a groove of positively-charged residues which wraps around the outside of the ring, unlike helicases, which typically thread DNA through the center of the ring
[32,41,43][32][41][43]. This domain has been shown to bind DNA with a footprint of 4 nucleotides per monomer, meaning that each heptameric ring could bind around 28 bp of DNA
[43,44,45][43][44][45]. RAD52 has also been shown to contain a secondary DNA-binding site that can interact with ssDNA, which would facilitate annealing of resected ends, as in SSA. This site can also bind double-stranded DNA (dsDNA), which enables strand exchange in an ATP-independent manner
[31,46][31][46]. Recent studies using atomic force spectroscopy have provided insights into the mechanics of RAD52/DNA interactions and illuminated possible molecular mechanisms for RAD52-facilitated annealing and strand exchange
[47]. In addition to canonical or “forward” strand exchange, where RAD52 binds ssDNA and facilitates its invasion of dsDNA, RAD52 can also perform inverse strand exchange by binding the duplex DNA, which is then invaded by ssDNA or RNA
[48,49][48][49].
3. RAD52 as a Target for Cancer Therapy
Despite its importance in a variety of repair pathways, RAD52 has long appeared dispensable for cell viability except in the context of BRCA-deficiency. This makes it an attractive therapeutic target in cancer, as tumors with BRCA mutations would be susceptible to loss of RAD52, while normal tissues with an intact HR pathway should survive without it. This genetic relationship is known as synthetic lethality, where loss of function of each gene on its own still results in viable cells, while loss of both results in cell killing. Synthetic lethality is an attractive strategy for targeted therapy, as it capitalizes on natural genetic relationships for tumor-specific kill instead of damaging agents, which can cause toxicity to both healthy cells and tumor cells
[21,87][21][50].
In the context of DNA repair, synthetic lethality can especially be exploited when tumor cells deficient in one repair pathway are “addicted” to a backup pathway. Loss of the backup pathway then leads to toxic intermediate structures and cell death. The most common example of pharmacological exploitation of synthetic lethality is poly-ADP-ribose polymerase (PARP) inhibition in HR-deficient cells. Since PARP can recognize single-stranded breaks (SSBs), inhibiting its function would leave unrepaired SSBs which could be converted to DSBs during replication. In HR-defective cells, these breaks would either be left unrepaired or would be channeled to error-prone repair processes. The first FDA-approved PARP inhibitor, olaparib, was authorized for treatment in a subset of BRCA-mutated cancers in 2014, followed by the development and approval of a new generation of inhibitors
[87][50]. While these inhibitors have been effective in some contexts, they have also been shown to cause global toxicity, likely because of the role of PARP proteins in cellular processes other than DNA repair
[88][51]. The effectiveness of PARP inhibitors supported the use of synthetic lethality as an indicator for therapeutic success, but their adverse effects have inspired some to look to other synthetically lethal interactions for the treatment of HR-deficient tumors.
As homozygous loss of RAD52 caused no effect in mice, it is unlikely to cause toxicity to healthy tissues in humans. Its effect in HR-deficient tumors, on the other hand, is significant and well-characterized. While initially described as being synthetically lethal with BRCA2, it has since been found to also lead to cell death in the absence of BRCA1, PALB2, and RAD51 paralogs
[20,21,89][20][21][52]. Additionally, recent super resolution microscopy data that mapped the spatiotemporal kinetics of HR repair showed that RAD52 and BRCA2 have distinct functions in the RAD51 nucleofilament formation and subsequent homology search. While in the absence of RAD52, BRCA2 was able to compensate for RAD52, the converse was not true
[90][53]. This may explain why RAD52 loss in a BRCA2-proficient context has no observable effect. In the context of BRCA1 loss, one proposed mechanism for synthetic lethality with RAD52 was reliant on the endonuclease EEPD1, which recognizes stalled forks and cleaves them into a DSB. In the absence of BRCA1 and RAD52, accumulation of unrepaired breaks at collapsed forks, which are normally repaired by HR, results in cell death. The synthetic lethality is rescued by EEPD1 co-depletion, as the forks are restarted or repaired by other mechanisms which, while error-prone, do not involve these toxic intermediates
[91][54].
Because RAD52 loss is so detrimental to BRCA-deficient cells, many groups have conducted screens to identify compounds that could serve as inhibitors of RAD52 function. The inhibitors target RAD52 in one of two ways: either by inhibiting its annealing activity by competitively binding to the ssDNA-binding domain, or by interfering with its ability to multimerize and form the ring structure required for its activity
[87,92,93,94,95,96][50][55][56][57][58][59]. Some of these compounds may be useful in HR-deficient cancers, either as monotherapy or in combination with other agents. One such inhibitor, 6-OH-dopa, was shown to have an additive effect on BRCA-deficient cancer cells treated with PARP inhibitors, which they termed “dual” synthetic lethality
[97][60]. Addition of RAD52 inhibitors could decrease the necessary dosage of PARP inhibitors, hopefully reducing their toxicity.
RAD52 inhibitors may also be useful outside the realm of BRCA deficiency, as depletion of RAD52 was shown to decrease tumor volume in FANCM-deficient tumors in mice. This interplay of RAD52 and FANCM was important in the repair and stability of common fragile sites, and relied on the translocase activity of FANCM rather than its association with the rest of the Fanconi anemia (FA) core complex
[98][61]. It remains to be determined whether any of the other myriad roles of RAD52 represent a new context in which an inhibitor may be useful.