Human Rad52 Facilitates a Three-Stranded Pairing that Follows No Strand Exchange: A Novel Pairing Function of the Protein Vasundhara M. Navadgi, Arnob Dutta, and Basuthkar J. Rao* Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai- 400 005, India ReceiVed June 17, 2003; ReVised Manuscript ReceiVed NoVember 1, 2003 ABSTRACT: Human Rad52 protein, by analogy with the genetics of yeast Rad52, is believed to mediate a pathway of homologous recombination even independent of Rad51. Current study is focused on unraveling the molecular properties of hRad52 that endow the protein such an ability. We show here that the hRad52 protein binds single-stranded DNA (ssDNA) as well as 3′- and 5′-tailed duplexes severalfold better than blunt-ended duplexes, altering the sensitivity of the bound DNA to the action of DNase I. Protein binding is sensitive to the length of the ssDNA: targets as short as a 33mer poorly bind the protein, whereas that of a 61mer and above bind the protein stably well. Such stable ssDNA-hRad52 complexes are highly competent in mediating not only the annealing of two complementary strands but also three-stranded pairing. The latter involves homologous recognition of linear duplex DNA by the ssDNA-hRad52 complex. We show that the hRad52 protein facilitates homologous recognition between ssDNA and duplex-DNA through a process that involves unwinding or transient unpairing of the interacting duplex via a novel three-stranded intermediate that does not lead to strand exchange. The results enable us to visualize a novel role for hRad52 that may model its function in a pathway requiring no hRad51. Cellular DNA is subjected to damage by a variety of reactive species inside the cell and external factors such as ionizing radiation and chemical mutagens, which give rise to double-strand breaks in the genome. The double-strand breaks also arise during normal cellular events such as DNA replication, meiotic recombination, and V(D)J rearrange- ments. These breaks, if left unrepaired, pose a serious threat to genomic integrity. Therefore, eukaryotic cells have evolved two robust distinct pathways to repair such breaks: a high fidelity homologous recombination (HR) 1 pathway and nonhomologous end-joining (NHEJ) pathway. The choice of the repair pathways appears to be regulated de- velopmentally, genetically, and during the cell cycle (1-3). Genes belonging to the RAD52 epistasis group are conserved in evolution and are shown to be involved mostly in homologous recombination events (4). It is now well- established that RAD51 and RAD52 constitute two important members of this group whose gene products exhibit ability to homologously pair DNA sequences. Saccharomyces cereVisiae Rad51 and human Rad51 are homologues of Escherichia coli RecA and promote homologous pairing and strand exchange reactions in vitro (1, 5). The RAD52 gene is conserved in yeast, humans, and mice but does not show any significant homology to the known recombination proteins in lower organisms. Despite the low sequence homology, the seven-membered ring structure of the Rad52 protein is remarkably similar to the oligomeric ring structures of E. coli RecT and -protein from bacteriophage λ, all of which are competent in facilitating DNA annealing (6-9). Thus, Rad52 is believed to be a functional homologue of RecT and the -protein. Moreover, RAD52 stands alone as the one gene required for all homologous recombination events, only some of which also require RAD51. In addition, RAD52 is involved in maintenance of telomeres in cells that lack essential components of yeast telomerase (4). A few recent studies point out that RAD52 in yeast is also involved in influencing NHEJ pathway events (10-12). Mechanistic basis of cooperation between various proteins belonging to the RAD52 epistasis group is becoming clear through various biochemical studies. Rad52 is shown to interact with Rad51 (13) and replication protein A (RPA) (14-16). Rad51 and Rad52 colocalize in distinct nuclear foci in response to DNA damage (17). Both yeast and human Rad52 stimulate Rad51 mediated strand exchanges (18-21). Rad52 is shown to displace RPA and assist the loading of Rad51 onto ssDNA with the help of the Rad55/Rad57 heterodimer (22-24). Rad52 mediated annealing of ssDNA is stimulated by RPA supposedly via specific protein-protein interactions (25, 26). In addition to strand annealing, hRad52 has been shown to promote homologous pairing between ssDNA and superhelical dsDNA substrates giving rise to D-loop formation (27-29). Recent hRad52 crystal structure of a conserved catalytic N-terminus domain revealed a 11-subunit oligomeric ring (29, 30) as opposed to a seven-subunit ring inferred from electron microscopic image reconstruction studies of full- length hRad52 protein (7). The crystal structure showed no basic amino acids in the central channel of the ring, but rather showed amino acids on the outside of the ring that forms a * To whom correspondence should be addressed. Fax: 91-22- 22804610, 22804611. Phone: 91-22-22804545 Ext. 2606. E-mail: bjrao@tifr.res.in. 1 Abbreviations: 2AP, 2-aminopurine; dsDNA, double-stranded DNA; DNase I, deoxyribonuclease I; hRad52, human Rad52; HR, homologous recombination; NHEJ, nonhomologous end joining; ssDNA, single-stranded DNA. 15237 Biochemistry 2003, 42, 15237-15251 10.1021/bi0350452 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/03/2003