The human Rad52 protein exists as a heptameric ring

Abstract

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

“…In counting more than 500 ringshaped oligomers for the 1-212 protein at concentrations of 1 and 4 M, we never observed the specific side-by-side associations as shown for wild type HsRad52 in Fig. 1, B and C. The observation of higher order oligomers for wild type HsRad52 is consistent with previous electron microscopic studies (7)(8)(9)(10)(11). Also, the lack of higher order oligomers for the 1-212 mutant is consistent with our previous identification of a new self-association domain in the C-terminal half of the protein that is responsible for the oligomerization of the 10-nm rings (11).…”

“…Both the yeast and human Rad52 proteins exist in heterogeneous oligomeric states including ring-shaped multimers ϳ10 nm in diameter, as well as higher order associations of these ringed structures (7)(8)(9)(10)(11). However, the functional relevance of these different oligomeric states has not been studied.…”

“…In either the absence or presence of DNA, Rad52 forms ring-shaped oligomers, ϳ10 nm in diameter, as well as higher order complexes of these rings (7)(8)(9)(10)(11). Rad52 binds to both single-and double-stranded DNA (7)(8)(9)(12)(13)(14)(15)(16), stimulates annealing of complementary DNA strands (7,(12)(13)(14)17), and promotes ligation of both cohesive and blunt-end fragments (9).…”

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

“…By contrast, the C-terminal segments of these RAD52 proteins contain the RAD51-and RPA-interaction regions and exhibit weak similarity Shen et al, 1996;Hays et al, 1998;Shinohara et al, 1998;Mer et al, 2000;Stasiak et al, 2000;Ranatunga et al, 2001). Moreover, the short forms of RAD52, resulting from alternative splicing, have been found to mostly contain the conserved N-terminal domain (Kito et al, 1999).…”

“…Its N-terminal is the most conserved domain across eukaryotic homologs Shen et al, 1996;Shinohara et al, 1997;Hays et al, 1998;Mer et al, 2000;Stasiak et al, 2000;Ranatunga et al, 2001). 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).…”