A radial way to anneal DNA

Reproduced with permission from National Academy of Sciences, USA, © 2002.

Eukaryotes have many tools to repair damaged DNA. One such tool — used to repair DNA double-strand breaks — is RAD52, which was discovered in yeast as a gene that helps protect against DNA damage caused by X-ray radiation, but that also repairs double-strand breaks during meiotic recombination. In vitro, the Rad52 protein binds to the Rad51 recombinase enzyme and stimulates its catalytic activity; Rad52 also interacts with single-stranded DNA (ssDNA) and induces annealing of complementary strands to form a duplex. But how does it promote DNA-duplex formation?

Wigley and colleagues now present the crystal structure of the ssDNA-binding amino-terminal domain (NTD) of Rad52. The NTD binds ssDNA in an unusual way, exposing every fourth nucleotide to attack by hydroxyl radicals in footprinting experiments. The structure reveals the basis for this repetitive pattern of DNA binding, and shows how Rad52 might promote the formation of double-stranded DNA (dsDNA).

The Rad52 NTD molecules are arranged as an undecameric ring, or wheel (see left-hand figure). Each Rad52 subunit has a core mixed α/β-fold domain that is connected to the carboxy-terminal helix by a flexible linker. The latter helix might be important in mediating Rad52's oligomeric state. The most striking feature of the Rad52 wheel, though, is a deep groove running along its entire outer rim (see right-hand figure). Amino acids at the bottom of this groove are positively charged (dark blue), whereas the residues that form the walls are hydrophobic. The groove is sufficiently wide to accommodate ssDNA, but appears to be too narrow for dsDNA (a ball-and-stick representation of ssDNA is modelled in the groove of the right-hand figure). The arrangement of amino acids indicates that the negatively charged DNA backbone might associate with the positively charged surface of the protein, leaving the DNA bases protruding out of the groove. Such an orientation might position the bases in such a way that allows the efficient formation of dsDNA. Further, the oligomeric nature of Rad52 might account for the four-base periodicity observed in footprinting experiments — the distance between each subunit in the Rad52 wheel is ∼22 Å, which could accommodate precisely four bases of extended ssDNA. Together, the structural characteristics of the Rad52 wheel indicate that two Rad52 oligomers might come together like interlocking cogs and, in doing so, bring together complementary strands of DNA and promote energetically favourable duplex formation.

It is interesting to note that the name RAD was coined as a geneticist's shorthand for radiation sensitivity. How appropriate then that it also means 'wheel' in German.

 REFERENCE Singleton, M. R. et al. Structure of the single-strand annealing domain of human RAD52 protein. Proc. Natl Acad. Sci. USA 21, 13492–13497 (2002)