Eukaryotic cells are exposed to both endogenous and exogenous insults to their genome. To foster genomic maintenance and protection, an elaborate DNA damage response (DDR) and DNA repair network evolved to encompass multiple repair pathways, each specializing in specific types of DNA lesions. Various sensor, mediator, and effector proteins are required to initiate and complete repair of damaged DNA. Deficiencies in essential components of the DDR or DNA repair pathways results in cell death or accumulation of mutations that can progress to cancer or other disease. For example, ataxia telangiectasia, ataxia-telangectasia-like disorder (AT-LD), Njimegen Breakage syndrome (NBS), and xeroderma pigmentosum are caused by mutations of ATM(1
), NBS1, and XPA respectively. In addition, mutations in the tumor suppressors genes BRCA1 and BRCA2 greatly increase susceptibility to breast, ovarian, and other cancers(3
One of the most threatening forms of DNA damage is the DNA double-strand break (DSB), as both strands of the DNA duplex are impaired simultaneously. The major repair pathways that cope with DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). In many organisms, Rad52 is a key protein involved in the HR pathway. This review will focus on Rad52, its role in HR and the translational opportunities available through understanding this protein and its function.
Homologous recombination pathways
Homologous recombination is the exchange of genetic information between allelic sequences and has an essential role in both meiosis and mitosis. In meiosis, HR allows for exchange of genetic material between paternal and maternal alleles within the gamete, and also coordinates proper segregation of homologous chromosome pairs during the first meiotic division.
In the somatic cell, HR maintains genomic integrity by promoting accurate repair of DSBs, damaged replication forks, and DNA interstrand crosslinks. HR repair requires a second, homologous DNA sequence to function as a donor template. In brief, the steps of DSB repair (DSBR) by HR are as follows: (a) recognition of a DSB, (b) processing of the DSB by nucleases to generate 3’ single-stranded DNA (ssDNA) tails, (c) formation of a Rad51 recombinase filament on ssDNA ends, (d) strand invasion into the homologous sequence with formation of the D-loop intermediate, (e) DNA polymerase extension from the 3’ end of the invading strand, (f) capture of the second end of the DSB by strand annealing after extension of the D-loop, (g) formation of two Holliday junctions (HJ), and (h) dissolution or resolution of the HJs to form crossover or noncrossover products (, left). Of note, the Rad51 recombinase is the essential enzyme that mediates strand invasion of a DNA duplex resulting in an exchange of DNA strands during HR.
Fig 1 The BRCA and RAD52 pathways of DNA double-strand break (DSB) repair. Various DNA lesions lead to DSBs that can be repaired by homologous recombination (HR) or single-strand annealing (SSA). A) In the HR pathway, after activation of the DNA damage response (more ...)
The synthesis-dependent strand-annealing (SDSA) pathway of HR differs from the DSBR pathway by diverging following step (e) and is not dependent on the second HJ formation. Rather, SDSA relies upon displacement of the invading strand and annealing it with the second resected DSB end, ultimately, producing a noncrossover product (, left).
When a DSB is flanked closely by sequence repeats, repair of the DSB may occur through a process called single-strand annealing (SSA). In SSA the DSB ends are resected, but rather than identifying a homologous DNA template for strand invasion, the resected strands anneal to one another using the repeat sequences for annealing. The result is a deletion of the sequence between the direct repeats (, right
). Since SSA is independent of strand invasion, the components involved in this process, such as Rad51 filament formation, and the downstream events, including HJ resolution, are not required(4
Single-ended DSBs can occur at telomeres or at broken replication forks. These can be repaired by HR through a single-ended invasion process known as break-induced replication (BIR)(5
). Most BIR events are dependent on Rad51 and other HR factors used in DSBR and SDSA. This process can also occur without Rad51 protein although the Rad51-independent process is thought to be only a minor component of this reaction.
A number of excellent reviews have been published with more in-depth discussion of HR and its sub-pathways (SSA, BIR)(4
Saccharomyces cerevisiae Rad52 protein and its function in mediating recombination
The S. cerevisiae
Rad52 protein (ScRad52) was identified in a genetic screen for mutants that are sensitive to ionizing radiation (IR)(8
) and is the most studied recombination mediator. Indeed, many other HR genes are labeled under the heading of RAD52
epistasis group, which includes RAD50
, and XRS2
. Of all members of the RAD52
epistasis group, the absence of RAD52
confers the most severe defects due to its involvement in all known pathways of HR, both RAD51
-dependent (DSBR, SDSA) and the RAD51
-independent pathway of SSA(6
Rad52 forms an oligomeric ring, and the oligomerization is mediated by the N-terminal portion of the protein(9
). This N-terminal portion specifically binds ssDNA(10
). ScRad52 mediates Rad51 strand invasion by physically associating with the Rad51 protein(11
) and allowing for highly efficient reversal of replication protein A (RPA)-imposed inhibition of the ssDNA-dependent ATPase and recombinase activity of Rad51(12
Rad52 also directly associates with RPA(15
), a hetero-trimeric ssDNA binding protein that coats the resected ends of the DSB (). Both the largest (RPA70) and intermediate (RPA32) subunits demonstrate direct binding with ScRad52(16
). The specific interaction of RPA and ScRad52 appears important for the recombination mediator function of ScRad52, as demonstrated when RPA is substituted with the Escherichia coli
single-stranded DNA-binding protein (SSB), ScRad52 is not able to overcome SSB-inhibition and subsequently fails to promote Rad51-mediated homology search and strand exchange(13
Additional functions of S.cerevisiae Rad52
ScRad52 is required for the Rad51-independent SSA and BIR reactions(4
). In concordance with its role in SSA, ScRad52 is able to anneal DNA strands that are either bare or coated with RPA(16
). There is some evidence to suggest that Rad52-mediated annealing of RPA-coated ssDNA strands is important for second ssDNA end capture in the DSBR pathway of HR(19
Human recombination mediators: Rad52 and BRCA2
Human Rad52 (hRad52) is similar to ScRad52 structurally and biochemically. hRad52 exists in an oligomeric form, binds ssDNA, promotes ssDNA annealing and, under certain specialized conditions, simulates Rad51-mediated homologous DNA pairing(20
). Like ScRad52, hRad52 has conserved the ability to directly interact with RPA(17
Subsequent studies have examined the N-terminal portion of hRad52 to determine in detail its interaction with ssDNA. Investigation by X-ray crystallography revealed an undecameric (11-subunit) ring structure with a deep groove on the surface that is lined by a vast number of positively charged basic and aromatic residues(21
). These hRad52 residues have been shown to be important for ssDNA binding by mutational analysis(22
) and deletions of equivalent residues in ScRad52 demonstrate deficiencies in DNA repair and HR(24
More recently, a second DNA binding domain has been discovered in hRad52, which appears to bind dsDNA(25
). Mutations of these amino acid residues are defective in promoting Rad51-mediated D-loop formation in ScRad52(26
). Further study of this second DNA binding domain is necessary to understand how it contributes to the known functions of Rad52.
Interesting, although hRad52 did not demonstrate recombination mediator activity in reconstituted biochemical assays(27
), it appears to mediate Rad51 function in human cancer cells deficient in BRCA1, PALB2
). We postulate that hRad52 may perform its mediator function in combination with partner proteins. It has been suggested that the Rad51 paralogs, Rad51B/C/D-XRCC2, may fulfill this role. However, the Rad51 paralogs appear to function primarily in the BRCA2 pathway and operate independently of hRad52 in mediating the Rad51 recombinase (Chun J and Powell SN, unpublished observations). There remains other Rad51 paralogs that may be candidate co-factors, including hSWS1 (homolog of yeast Shu2) and SWSAP1(30
). Alternatively, hRad52 recombination mediator activity may be dependent on specific posttranslational modifications (e.g. phosphorylation, SUMOylation, etc.) that have yet to be examined biochemically.
Besides its role in Rad51 mediation, it has recently been suggested that BRCA2 participates in the stabilization of RAD51 filaments from degradation by MRE11 through interaction of BRCA2’s conserved C-terminal domain with Rad51(31
). It is unknown whether Rad52 has any role to play in this proposed mechanism of replication fork protection.
Species spectrum of Rad52 and BRCA2
BRCA2 and its homologs appear to have appropriated subsets of Rad52 function, especially in mediating recombination by Rad51, and are present in a variety of multi-cellular organisms(See )(32
The spectrum of BRCA2 and RAD52 across various species.
A speculative assumption is that BRCA2 has evolved for the greater level of insults to the genome in multi-cellular organisms in comparison to unicellular organisms. Seen from another perspective, increased -- but regulated -- mutagenesis is evolutionarily preferential in unicellular life; however in multi-cellular organisms more stringent control of DNA repair, especially in stem cells, via BRCA2 is essential for vitality. U. maydis exists as a unique unicellular exception to possessing a BRCA2 homolog, possibly, to enable it to tolerate the greater levels of exogenous DNA damage (e.g. high UV exposure, etc.) it encounters in its native environment budding on crops of corn or in support of its multi-cellular filamentous form.
Rad52 and its synthetically lethal interactions across various species
The dual role of ScRad52 in both mediating Rad51 function and performing the annealing step in second end capture and SDSA explains why Sc
mutants display more severe phenotypes than defects in Rad51 protein. Unexpectedly, in organisms containing a BRCA2 homolog, including U. maydis
, chicken, and mice, inactivation of Rad52 causes minimal or no HR and DNA repair defects(39
). However, in human cancer cell-lines deficient in BRCA1, PALB2
depletion increases damage-induced chromosomal abnormalities, decreases clonogenic survival, and further reduces the rates or frequency of HR(28
). Thus, RAD52
appears to exist in a relationship with BRCA1
, or BRCA2
, known as synthetic lethality,
where simultaneous inactivation of two genes leads to cell death, whereas inactivation of only one of these genes does not affect viability. This observation is in accordance with other RAD52
synthetically lethal phenotypes seen in chicken DT40 cells, where inactivation of rad52
is lethal with a defect in XRCC3
, a RAD51 paralog(42
), and, in U. maydis
, where a Rad51 paralog mutant, rec2
, demonstrates synthetic lethality with loss of rad52
Interestingly, in DT40 cells, rad52
deletion in a BRCA2
mutant background did not enhance cytotoxicity, instead epistasis was observed between BRCA2 and Rad52, as well as other HR proteins(43
). Also, in U. maydis, Brh2
mutants in combination with Rad52
defects demonstrate a more subtle synthetically lethal phenotype, which the study’s authors interpret as a compensatory interaction(39
). These observed divergences from human and other studied organisms will require further investigation to delineate the hierarchy and functional nuances of these various HR proteins across the evolutionary spectrum.
Dependency of BRCA1-PALB2-BRCA2 deficient human tumor cells on Rad52-Rad51-mediated HR
BRCA1, PALB2 and BRCA2 appear to be linked in a sequential manner. BRCA2 recruitment to foci requires interaction with PALB2, and abolishment of the physical interface between BRCA1 and PALB2 impairs BRCA2 function and subsequently Rad51-mediated HR(44
). Evidence suggests Rad52 provides an alternative mediator pathway to the BRCA1-PALB2-BRCA2 pathway and allows tumor cells to proliferate in the absence of the BRCA pathway (). The loss of Rad52 in a BRCA1, PALB2 (28
), or BRCA2(29
) mutant leads to cell death.
These observations set the potential foundation for any BRCA1-PALB2-BRCA2 pathway deficient tumor to be therapeutically targeted by Rad52 inactivation. This approach for targeting BRCA pathway deficient cancers is distinct from other strategies that take advantage of synthetic lethality in BRCA mutant tumors, namely poly-(ADP-ribose) polymerase (PARP) inhibition. These translational approaches and discoveries that exploit HR defective cancers will be highlighted in the subsequent section.