The Rad52 protein, which is unique to eukaryotes, plays important roles in the Rad51-dependent and the Rad51-independent pathways of DNA recombination. In the present study, we have biochemically characterized the homologous pairing activity of the HsRad52 protein (Homo sapiens Rad52) and found that the presynaptic complex formation with ssDNA is essential in its catalysis of homologous pairing. We have identified an N-terminal fragment (amino acid residues 1-237, HsRad52 1-237 ) that is defective in binding to the human Rad51 protein, which catalyzed homologous pairing as efficiently as the wild type HsRad52. Electron microscopic visualization revealed that HsRad52 and HsRad52 1-237 both formed nucleoprotein filaments with single-stranded DNA. These lines of evidence suggest the role of HsRad52 in the homologous pairing step of the Rad51-independent recombination pathway. Our results reveal the striking similarity between HsRad52 and the Escherichia coli RecT protein, which functions in a RecA-independent recombination pathway.

The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C͞Rad51L2. The purified Xrcc3⅐Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3⅐Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3⅐Rad51C. Electron microscopic observations revealed that Xrcc3⅐Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

Rad52 plays essential roles in homology-dependent doublestrand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.The repair of DSBs 4 is a critical process by which cells maintain genome integrity. In eukaryotic cells these breaks are repaired by homologous recombination (HR) and non-homologous end joining. In the homologous end joining pathway, broken DNA ends are rejoined by religation or annealing of short common sequences (microhomologies) near their ends.Because homologous end joining lacks a mechanism to reject erroneous pairings that occur by chance, recessed DNA, chromosome rearrangements, and mutations are possible outcomes of this repair pathway (1). By contrast, the HR pathway achieves high accuracy in DSB repair by utilizing mechanisms for a DNA homology search. In this pathway the ends of the DSBs are resected to generate 3Ј single-stranded tails. The singlestranded region then either invades an undamaged homologous duplex DNA (strand invasion) or pairs with a complementary single strand (single-strand annealing). The importance of this pathway is underscored by the high conservation, from yeast to humans, of the factors that catalyze HR (2).Rad52 is one of the key players in the yeast HR pathway, and its homologs have been found in higher eukaryotes. In yeast, Rad52 is essential in both the RAD51-dependent and -independent HR pathways and functions in several homology-dependent DSB repair events, including gene conversion, breakinduced replication, and recombination between inverted repeats (3). Several biochemical studies have established that Rad52 functions as a mediator in RAD51-dependent HR pathways. These findings include facilitating the assembly of the Rad51 recombinase on replication protein A-coated ssDNA (4, 5) and stimulating the DNA strand exchange activity of the Rad51 recombinase (6 -9). In vitro studies have also suggested more direct roles of Rad52 in HR. Rad52 catalyzes the annealing of complementary ssDNA and promotes D-loop formation and DNA strand exchange (10 -14). The strand invasion promoted by Rad52 may be relevant to some RAD51-independent HR events such as break-induced replication, in...

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270 -328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in doublestranded DNA binding and that they constitute a part of a gateway for homologous recognition.RecA protein is a key enzyme in homologous genetic recombination and recombinational repair of damaged DNA. RecA protein, which consists of 352 amino acid residues, promotes the formation of heteroduplex joints between single-stranded and double-stranded DNA by homologous pairing and strand exchange in vitro (1, 2). In homologous pairing, RecA protein first binds to single-stranded DNA in the presence of ATP ("primary binding"; Ref.3) and forms helical nucleoprotein filaments called "presynaptic filaments," in which singlestranded DNA is extended to a length 1.5 times longer than the B form DNA (4 -7). Then, presynaptic filaments bind to doublestranded DNA ("secondary binding") and form a three-component complex including single-stranded DNA, double-stranded DNA, and RecA protein (and ATP) without prior homologous alignment. The search for homology between single-stranded and double-stranded DNA occurs in this three-component complex (3,8,9).We had shown that a chimeric RecA protein of Escherichia coli and Pseudomonas aeruginosa, RecAc38, is defective in homologous pairing of single-stranded DNA with double-