RAD52, the primary single strand annealing protein (SSAP) in humans, forms undecameric rings that bind ssDNA within a positively charged groove. Current models suggest RAD52 may either anneal complementary ssDNAs on the same ring in cis, or between two rings loaded with ssDNAs in trans. However, there are currently no structures of RAD52 bound with multiple strands, leaving its annealing mechanism unknown. To understand common features of SSAPs, we turn to a distant RAD52 homolog from yeast called Mgm101. Mgm101 is essential for mitochondrial DNA (mtDNA) replication and repair of double-stranded DNA breaks caused by oxidative damage. We determined cryo-EM structures of Mgm101 alone and in complexes with ssDNA, with a duplex intermediate of annealing, and with B-form dsDNA product. In all states, Mgm101 forms a closed nonadecameric (19-mer) ring that binds the backbone of the first ssDNA at the base of a narrow groove. The second complementary strand binds directly on top of the first to form an extended, unwound, and circular duplex intermediate of annealing. A fourth structure suggests the annealed duplex product leaves the groove, assumes a B-form conformation, and associates with a novel β-hairpin motif located at the upper and outer rim of the Mgm101 ring. Mass photometry and native mass spectrometry suggest the complexes observed by cryo-EM are also present in solution. Altogether, our data reveal four structural snapshots of the Mgm101 single-strand annealing pathway to suggest it anneals two complementary ssDNAs on the same ring in cis. As the ring-shaped architecture and core DNA-binding elements are conserved in RAD52 and related proteins, they are likely to use a similar cis mechanism of annealing.
Wheat et al. (Sun,) studied this question.