Identification of a second DNA binding site in the human Rad52 protein

Abstract

Rad52 plays essential roles in homology-dependent double-strand 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.

FIGURE 1.

Alanine scan mutagenesis of Rad52^1-212. A, EMSA. Alanine point mutants (0, 0.5, 1, 1.5, or 2 μm) of Rad52^1-212 were incubated with 15 μm negatively supercoiled plasmid DNA (pGsat4, 3.2 kilobases), and complexes were fractionated through an agarose gel. WT, wild type. B, surface view of the Rad52^1-212 protomer. The stem and domed cap regions are colored light brown and light blue, respectively. Lys-102 and Lys-133 (magenta) are located at the rim of the stem region, whereas Lys-169, Tyr-171, and Arg-173 (also magenta) are located at the edge of the domed cap region. The previously identified ssDNA binding residues (Arg-55, Tyr-65, Lys-152, Arg-153, Arg-156, dark blue) are clustered at the bottom of the groove formed between the stem and domed cap regions. C, surface view of the Rad52^1-212 undecameric ring. All structural figures were created using the PyMOL program.

FIGURE 2.

D-loop formation activities of Rad52^1-212 mutants. A, a schematic diagram of the D-loop formation reaction promoted by Rad52^1-212. B, increasing concentrations (0, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2 μm) of wild-type (WT) or mutant Rad52^1-212 proteins were initially complexed with 1 μm ssDNA (5S-repeat-10^-1, 50-mer) (first row). Afterward, negatively supercoiled plasmid DNA (pB5Sarray, 5.3 kilobases) was added, and the reactions were either treated with glutaraldehyde (ternary complexes, second row) or proteinase K (D-loops, third row).

FIGURE 3.

ssDNA annealing activities of Rad52^1-212 mutants. A, a schematic diagram of the ssDNA annealing reaction promoted by Rad52^1-212. B, percentages of DNA annealing catalyzed by the wild-type (WT) Rad52^1-212 (closed square), K102A (open diamond), K133A (open triangle), and K102A/K133A (open square) proteins. Proteins (0.25 μm) were first complexed with 1 μm ssDNA (sense SAT-1-50, 50-mer) followed by the addition of a complementary ssDNA (antisense SAT-1-50, 50-mer). The spontaneous reaction is indicated by closed circles.

FIGURE 4.

Electron micrographs of Rad52^1-212 complexed with covalently closed, relaxed plasmid DNA. Negatively stained samples of Rad52^1-212, the Rad52^1-212-DNA complex, and the Rad52^1-212 K102A-DNA complex (from left to right) are shown. The black bars denote 10 nm.

FIGURE 5.

Positive supercoiling activity of Rad52 and Rad52^1-212. A, schematic diagram of the topoisomerase I-mediated relaxation assay. B and C, one-dimensional gel electrophoresis of DNA topoisomers generated in the presence of Rad52 (B) and Rad52^1-212 (C). In these reactions the protein concentrations were increased from 0 to 4 μm in 0.5 μm increments. ccrDNA denotes covalently closed, relaxed DNA. D, two-dimensional gel electrophoresis of DNA topoisomers generated in the presence of Rad52 and Rad52^1-212. The 2 μm Rad52 reaction (B, lane 6), resolved in a two-dimensional gel, is shown in D, top right. Similarly, the 2 μm Rad52^1-212 reaction (C, lane 6), resolved in a two-dimensional gel, is shown in D, bottom right. The top left figure in D is a schematic diagram of the relative positions of covalently closed, relaxed DNA (ccr), nicked circular DNA (nc), positively supercoiled topoisomers (i), and negatively supercoiled topoisomers (ii) resolved by two-dimensional gel electrophoresis. E, alanine scan mutagenesis of Rad52^1-212. The mutant proteins (0, 0.5, 1, 1.5, or 2 μm) were incubated with 30 μm of covalently closed, relaxed plasmid DNA. CQ, chloroquine.

FIGURE 6.

DNA minor groove binders inhibit the positive supercoiling activity of Rad52^1-212. A, structures of distamycin A, netropsin, and pentamidine. B, topoisomerase I-mediated relaxation assay in the presence of minor groove binding ligands. Increasing concentrations of the ligands with or without 2 μm Rad52^1-212. Lanes 12, 24, and 36 show the addition of the ligands before E. coli topoisomerase I. C, DNA binding activity of Rad52^1-212 in the presence of the ligands. Negatively supercoiled plasmid DNA (pGsat4, 3.2 kilobases) was used.

FIGURE 7.

A ternary complex model depicting the first and second DNA binding sites on Rad52^1-212. The Lys-102 and Lys-133 residues are colored magenta. The ssDNA molecule (yellow) binds inside the groove, whereas the dsDNA molecule (orange) runs around the rim of the stem region. The DNA model was created using the NAB program (40), with ΔLk =+2. Lk, linking number.

FIGURE 8.

A unifying view of the ssDNA annealing and strand invasion reactions promoted by Rad52. For simplification, a Rad52 protomer (blue) is shown. Both ssDNA annealing and D-loop formation require the initial formation of the Rad52-ssDNA complex. The second DNA substrate then binds to the second DNA binding site, and the reactions take place at the entrance of the groove.