RAD52 Protein: Structure and Functional Modifications

During the IVth International Yeast Genetics Conference in 1970, it was proposed that all genetic loci, which mutations confer to X-ray sensitivity, would be given the name “rad” followed by an identification number. Rad52 was first established four years later in S. cerevisiae, where its mutation resulted in the abolishment of all recovery processes after irradiation with X-rays.

The crystal structure of purified human RAD52 has suggested it forms a ring-shaped undecamer; however, biophysical studies show that RAD52 in vitro could exist as a heptameric ring with a positively charged ssDNA-binding groove running around the structure (Figure 1). DNA-binding domains have, however, been found on both C- and N-terminal parts of the molecule, therefore supporting speculation of another binding-region outside the first groove of the protein oligomer. RAD52 also contains a residue responsible for its import into the nucleus (nuclear localization signal = NLS) which, in human RAD52, is located at the C-terminal end of the protein. The RAD52 NLS is weak when the protein is in monomeric form and allows for only slow migration into the nucleus; however, in the oligomeric ring structure, the additive effect of seven NLS would allow for more efficient transport to the nucleus. For this reason, the most likely formation of RAD52 heptamer is occurring in the cytoplasm. The N-terminal domain of RAD52 allows for its heptamerization, and it possesses the ability to interact with RAD59. The RAD52/RAD59-dependent recombination pathway appears to be important for the processing of faulty Okazaki fragments. The C-terminal and the central domains of RAD52 facilitate recombination “mediatory function” of the protein.

Figure 1. Human RAD52 structure, post-translational modifications, and functions. N-terminal fragment of RAD52 contains a region responsible for its oligomerization and binding with DNA molecule. C-terminal area includes domains interacting with replication protein A (RPA) and RAD51 recombinase, as well as nuclear localization signal (NLS) region responsible for RAD52 transportation to the nucleus. According to the “nuclear retention model”, RAD52 monomer possesses a weak NLS signal allowing only slow transport to the nucleus where RAD52 undergoes oligomerization. The “additive NLS model” suggests formation of RAD52 ring in the cytoplasm, resulting in an additive NLS effect and more robust RAD52 ring transportation to the nucleus. Activity of RAD52 and its participation in different recombination processes can be modulated by post-translational modifications including SUMOylation, phosphorylation, and acetylation.

A variety of post-translational modifications including acetylation, phosphorylation, or (SUMO)ylation modulates the function of numerous proteins. The involvement of human RAD52 in HR repair depends on its acetylation by histone acetyltransferases (HATs) p300/CBP. Unacetylated RAD52 dissociates from DSB along with RAD51 recombinase. The acetylation status of RAD52 is maintained by continuous cooperation between HATs and the histone deacetylases (HDACs) sirtuin2 (SIRT2) and SIRT3. RAD52 can also undergo (SUMO)ylation which does not influence its protein–protein interactions, although it delays recombination by inhibition of DNA-binding and strand annealing activities. SUMO modification also sustains the activity of yeast Rad52 and protects it from degradation. Phosphorylation of RAD52 by c-ABL1 kinase at tyrosine 104 seems to enhance ssDNA annealing activity and inhibit dsDNA binding abilities of RAD52. Constitutively active oncogenic BCR-ABL1 kinase facilitates nuclear localization of RAD52 and stimulates SSA repair in leukemia cells.

Role of RAD52 in DNA Repair

RAD52 is able to bind ssDNA, facilitating a major role in single strand annealing (SSA) and HR repair of DSBs based on the homologous strand. RAD52 can also operate on single-ended DSBs, preventing excessive degradation of stalled replication fork by converting them into a compact conformation that is less available for reversal enzymes. In checkpoint-deficient cells, RAD52 reverses stalled replication forks to the form in which they can be cleaved by the MUS81/EME1 complex during the process of break-induced replication (BIR). In fact, RAD52, through its ssDNA annealing activity, is suspected to assemble a displacement loop (D-loop) which invades the homologous chromosome and allows for BIR progression on the template of the homologous sequence. RAD52 can also prevent chromosome end exposure by copying telomere caps from other chromosomes in a subtype of HR—alternative lengthening of telomeres (ALT).

Homologous Recombination (HR)

Although in S. cerevisae RAD52 is a predominant recombination protein acting alone in facilitating RAD51 loading onto ssDNA, in mammals its role seems to be diminished by other proteins, namely BRCA1/2. Rad52-/- mice are viable, fertile, and show only a slight decrease in HR activity. However, overexpression of RAD52 in mammalian cells enhanced their resistance to ionizing radiation, indicating the importance of RAD52 in the DNA damage response. It has been demonstrated that in the absence of the BRCA1/2-dependent HR pathway, cell viability may be dependent on RAD52-RAD51, indicating that in mammalian cells, HR operates with at least two alternative sub-pathways: BRCA1/2-dependent canonical mechanism and RAD52-dependent alternative repair. In the latter, RAD52 interacts with RAD51 and places it on RPA-coated ssDNA overhangs, which is possible thanks to the strong inhibitory effect that RAD52 exerts on the RPA-ssDNA complex. It has been suggested that once RAD51 is localized at the DSB, most of RPA and RAD52 are displaced from the DNA; however, some persist surrounded by recombinase filaments, possibly stabilizing further steps of HR.

Single Strand Annealing (SSA)

In general, DSB end resection and the creation of single-stranded overhangs is a pivotal moment of DNA repair which allows for the cell to choose between not only NHEJ and HR, but also between HR and SSA depending on how far resection has proceeded. SSA events require sufficient resection to have direct sequence repeats presented in the form of ssDNA. 53BP1 is a factor that is responsible for the suppression of BRCA1-mediated end resection and the promotion of D-NHEJ. It has been suggested that the absence of 53BP1 leads to hyper-resection of DSBs in G2/S phase. This stage leads to the switch from error-free HR to mutagenic RAD52-mediated SSA. Therefore, cells lacking BRCA1 and 53BP1 require RAD52 for the maintenance of DSBs. In addition, other factors—BRCA1, RNF168, RIF1, histone H2A.X—which inhibit end resection, were shown to suppress SSA.

In SSA, 5’ to 3’ end resection within tandem repeats exposes about 25 nt ssDNA overhangs. RAD52 interacts with RPA-coated overhangs and aligns the complementary regions. It was suggested that after finding initial homology, a further search for stronger interactions and more extensive homology continues without complex dissociation. The alignment occurs due to the overlapping of nucleoproteins present on the opposite sites of DSB. After final homology is achieved, the endonucleolytic complex ERCC1/XPF, in cooperation with RAD52, trims 3’ overhangs. Final gap filling and strand ligation follow this step. SSA often results in the generation of deletions during the step where 3’ ssDNA overhangs are trimmed. Additionally, since SSA uses as templates repetitive elements that are present in multiple other genetic loci, SSA may also lead to translocations.

RNA-Dependent DNA Recombination

Although HR is mostly active during G2/S phase due to the short proximity to homologous sequence of sister chromatid or homologous chromosome, it appears that a HR sub-pathway that uses RNA transcripts as a template is active at transcriptionally active regions during G1/G0 phase of the cell cycle. RNA polymerase II can bypass different base modifications, however single strand breaks (SSBs) and DSBs result in permanent blockage of the enzyme. Such damage in transcriptionally active regions is expected to be more toxic than in any other genome area.

Under conditions of low abundance of BRCA1/2 during G0 and early G1 phase, its task of RAD51 recruitment to a DSB is fulfilled by RAD52. It appears that RAD52 may not only show affinity to ssDNA but also to RNA, and it might be active in repair mechanisms in differentiated, non-dividing cells. Cocaine syndrome B protein (CSB) is expected to be the key protein in transcription-coupled homologous recombination (TC-HR). It detects stalled RNA polymerase and interacts directly with HR proteins RAD51C and RAD52, directing them to DNA damage in coding regions. RAD52 binds to R-loops, which are three stranded DNA-RNA hybrids that allow for repair on the template of RNA transcript. In transcription-associated homologous recombination repair (TA-HR) RAD52 is recruited to the RNA-DNA hybrid at the DSB and promotes ERCC excision repair 5 (XPG)-mediated processing, leading to HR-based repair.

Two models indicate how RAD52 might promote RNA-mediated repair. In the first, RAD52 directs RNA to the DSB, where it finds homologous sequences with both its termini, creating a synapse that conjoins the ends. In the second model, RAD52 creates an RNA-DNA hybrid at 3’ ssDNA overhang. The overhang created by RNA is then used as a template for reverse transcription, before finally being degraded by RNase H. In the final steps, homology between the created ssDNA and the other end of the DSB allows for end joining and RAD52-promoted SSA.

It has been established that not only yeast, but also human RAD52 promotes RNA-templated DNA repair. RNA could constitute a stable template for DSB repair in differentiated cells that do not undergo divisions thus do not have sister chromatid as a template.