Structure of the single-strand annealing domain of human RAD52 protein - PubMed (original) (raw)

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

Martin R Singleton et al. Proc Natl Acad Sci U S A. 2002.

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 single-strand annealing in vitro. We have determined the crystal structure of this single-strand annealing domain at 2.7 A. 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.

PubMed Disclaimer

Figures

Fig 1.

Fig 1.

Biochemical activities of RAD521–209. (A) Purification of His-tagged RAD521–209. Recombinant protein was overexpressed in E. coli and purified by chromatography on Talon, affi-gel heparin, and ssDNA cellulose as described in Experimental Procedures. Lane M, molecular mass markers. (B) Annealing of complementary ssDNAs by RAD521–209. Reactions contained two complementary oligonucleotides, of which one was 32P-labeled. The products were analyzed by neutral PAGE as described in Experimental Procedures. dsDNA, double-stranded DNA. (C) RAD521–209 is defective in its ability to stimulate recombination reactions promoted by RAD51. Reactions containing single-stranded circular and linear duplex φX174 DNA were supplemented with the indicated proteins as described in Experimental Procedures, and the products of incubation were analyzed by agarose-gel electrophoresis. The DNA was visualized by staining with SYBR green and PhosphorImaging. The positions of joint molecules, linear duplex, and nicked circular-DNA products were determined by comparison with DNA markers (not shown).

Fig 2.

Fig 2.

Hypersensitivity of ssDNA bound by RAD521–209 to hydroxyl radicals. 5′-32P-labeled poly(dT)40 was incubated with the indicated concentrations of RAD52 or RAD521–209, and the products were probed with hydroxyl radicals as described in Experimental Procedures. 32P-labeled products were analyzed by denaturing PAGE and autoradiography.

Fig 3.

Fig 3.

Structure of the RAD521–209 undecamer. (A) Overall fold of the protein monomer. Domain 1 (residues 24–177) is colored blue, and domain 2 (residues 178–209) is colored red. (B) The undecamer viewed along the 11-fold symmetry axis. (C) Stereo image of the subunit contacts viewed at 90° from the view in B. Three adjacent subunits are shown in yellow, red, and blue to illustrate the subunit contacts. The remainder of the undecamer is shown in gray. This figure (and Figs. 4 and 5) was prepared by using PYMOL (

http://pymol.sourceforge.net

).

Fig 4.

Fig 4.

Molecular surface of the protein. (A) Electrostatics of the molecular surface of the protein as determined in GRASP (30). Negative potential is shown in red, and positive potential is shown in blue. A deep groove that runs across the protein surface suggests a possible binding site for DNA. Modeled ssDNA is overlaid for reference. (B) A ribbon representation of the undecamer with modeled DNA (in cyan). Residues proposed to be involved in binding to DNA (residues 39–80) are shown in red with the remainder of the molecule shown in gray. The molecular boundary is shadowed in pale gray.

Fig 5.

Fig 5.

ssDNA-binding site. Ribbon representation of the proposed ssDNA-binding site spanning two adjacent subunits. Residues 39–80 of the protein are colored red with the remainder shown in gray. The ssDNA is overlaid in cyan.

Similar articles

Cited by

References

    1. Game J. C. (1983) in Yeast Genetics: Fundamental and Applied Aspects, eds. Spencer, J. F. T., Spencer, D. H. & Smith, A. R. W. (Springer, New York), pp. 109–137.
    1. Petes T. D., Malone, R. E. & Symington, L. S., (1991) The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis and Energetics (Cold Spring Harbor Lab. Press, Plainview, NY), Vol. 1, pp. 407–521.
    1. Mortensen U. H., Bendixen, C., Sunjevaric, I. & Rothstein, R. (1996) Proc. Natl. Acad. Sci. USA 93, 10729-10734. - PMC - PubMed
    1. Shinohara A., Shinohara, M., Ohta, T., Matsuda, S. & Ogawa, T. (1998) Genes Cells 3, 145-156. - PubMed
    1. Sugiyama T., New, J. H. & Kowalczykowski, S. C. (1998) Proc. Natl. Acad. Sci. USA 95, 6049-6054. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources