Structure of the single-strand annealing domain of human RAD52 protein

Abstract

In eukaryotic cells, RAD52 protein plays a central role in genetic recombination and DNA repair by (i) promoting the annealing of complementary single-stranded DNA and (ii) stimulation of the RAD51 recombinase. The single-strand annealing domain resides in the N-terminal region of the protein and is highly conserved, whereas the nonconserved RAD51-interaction domain is located in the C-terminal region. An N-terminal fragment of human RAD52 (residues 1-209) has been purified to homogeneity and, similar to the full-size protein (residues 1-418), shown to promote singlestrand annealing in vitro. We have determined the crystal structure of this single-strand annealing domain at 2.7 Å. The structure reveals an undecameric (11) subunit ring with extensive subunit contacts. A large, positively charged groove runs along the surface of the ring, readily suggesting a mechanism by which RAD52 presents the single strand for reannealing with complementary single-stranded DNA.recombination ͉ DNA repair ͉ crystallography T he ability of a cell to survive agents that promote genome breakage requires efficient recombinational repair systems. In lower eukaryotes, repair involves the RAD52 epistasis group of genes including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 (1,2). A key member of this group, RAD52, encodes a protein that plays a dual role in recombination by (i) promoting the annealing of complementary single strands (3-5) and (ii) stimulating in vitro recombination reactions catalyzed by the RAD51 recombinase (6-8).Human RAD52 protein shares many properties with its yeast counterpart including DNA strand annealing and RAD51 stimulation activities (9-11). The human protein has been visualized by electron microscopy, and a low-resolution three-dimensional structure has been determined (12, 13). Human RAD52 consists of seven subunits that are organized in the form of a ring with a large central channel. The Saccharomyces cerevisiae RAD52 protein has been shown also to form rings, but no detailed structure is available presently (4).The mechanism by which RAD52 promotes single-stranded DNA (ssDNA) annealing is unknown. However, when complexes formed between RAD52 and ssDNA were probed with hydroxyl radicals, a unique repeating four-nucleotide hypersensitivity pattern was observed (14). Sequence-independent hypersensitivity was observed over Ϸ36 nucleotides and was phased precisely from the terminal nucleotide. These results led to the proposal that RAD52 binds ssDNA via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein ring.Sequence comparisons, site-directed mutagenesis, and biochemical studies indicate that the highly conserved N-terminal region of RAD52 possesses ssDNA annealing activities, whereas the RAD51-interaction domain is located toward the nonconserved C-terminal region (15-18). Furthermore, the RAD59 protein shares sequence homology with the N-terminal region of RAD5...

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

“…Moreover, an unusual nonglobular tower domain interrupts one of the BRCA2 OB folds (16) and may act to promote BRCA2 binding to dsDNA-ssDNA junctions (39). Consistent with a bypass mechanism, we found that the structurally distinct ssDNA-binding domain from Rad52 (40,41) can function in HDR correction in the Brca2 mutant cells. Besides the DNA-binding domain, other parts of the BRCA2 protein may provide more complex levels of control of BRCA2 function, including the recently identified cell cycle-regulated phosphorylation site that modulates Rad51 binding to the C terminus of BRCA2 (17).…”

Identification of PlantRAD52Homologs and Characterization of theArabidopsis thaliana RAD52-Like Genes

“…The described sequence similarity allowed us to model the structure of plant RAD52 homologs on the determined structures of the N-terminal domain of human RAD52 (Kagawa et al, 2002;Singleton et al, 2002). As expected by the relatively long corresponding regions and few insertion/deletion points ( Figure 1; see Supplemental Figure 1 online), the same topology and structure features of human RAD52 are present in the models of A. thaliana RAD52 homologs (see Supplemental Figure 2 online).…”

“…As expected by the relatively long corresponding regions and few insertion/deletion points ( Figure 1; see Supplemental Figure 1 online), the same topology and structure features of human RAD52 are present in the models of A. thaliana RAD52 homologs (see Supplemental Figure 2 online). The residues corresponding to the two known RAD52 DNA binding sites (Kagawa et al, 2002;Singleton et al, 2002) are mostly conserved in the plant RAD52 homologs (Figure 1) and form similar sites, including both the positively charged groove and the second DNA binding site (see Supplemental Figures 2B and 2C online). The alpha helix of the RAD52 stem region (residues 145 to 163 in At1g71310, corresponding to residues 159 to 134 in human RAD52) demonstrated a repetitive pattern of sequence conservation.…”

“…This domain enables the formation of a ring-shaped oligomer by including sites for selfassociation and in yeast for interaction with its RAD59 paralog (Shinohara et al, 1998;Stasiak et al, 2000;Ranatunga et al, 2001). The external part of the ring formed in the human homolog was shown to include a positively charged, DNA binding groove (Kagawa et al, 2002;Singleton et al, 2002). The central portion contains a domain for interaction with RPA (Hays et al, 1998).…”

Loss of Rad52 partially rescues tumorigenesis and T-cell maturation in Atm-deficient mice

“…Rad52 repair foci colocalize with DSBs in vivo in yeast with one foci recruiting multiple DSBs (Lisby et al, 2003). Furthermore, the structure of the single-strand annealing domain of human Rad52 was shown to form an 11-subunit ring (Kagawa et al, 2002;Singleton et al, 2002). The Rad54 protein cooperates with Rad51 in the homologous DNA pairing reaction and is involved in the dissociation of Rad51 from nucleoprotein complexes formed on dsDNA (Sigurdsson et al, 2002;Solinger et al, 2002).…”