Poster abstracts
Poster number 55 submitted by Carter Wheat
Cryo-EM structures of yeast RAD52 homolog Mgm101 in complex with DNA reveal snapshots of a full single-strand annealing pathway
Carter T Wheat (Biological Chemistry and Pharmacology, The Ohio State Biochemistry program), Zihao Qi (Department of Chemistry and Biochemistry), Miqdad Hussain (Biological Chemistry and Pharmacology), Vicki Wysocki (School of Chemistry and Biochemistry, Georgia Institute of Technology), Charles E. Bell (Biological Chemistry and Pharmacology)
Abstract:
Double-stranded DNA break (DSB) repair by single-strand annealing (SSA) can occur when directly repeated sequences flank the DSB. Once the 5’ ends of the break are resected by exonucleases, single-strand annealing proteins (SSAPs) bind the resulting 3’ overhang and anneal complementary strands. The primary SSAP in humans, RAD52, forms undecameric rings that bind ssDNA within a positively charged groove. Current models suggest RAD52 may either anneal complementary ssDNAs in cis (on the same ring), or in trans, between two rings loaded with ssDNAs. 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 Mitochondrial Genome Maintenance protein 101 (Mgm101). Mgm101 is essential for mitochondrial DNA (mtDNA) replication, and repair of DSBs caused by oxidative damage. We have solved Cryo-EM structures of Mgm101 bound to ssDNA substrate or to annealed duplex intermediate. The Mgm101-ssDNA structure forms a nonadecameric (19-mer) ring with ssDNA bound in an extended conformation in the groove with its nucleobases facing outward, reminiscent of prior RAD52-ssDNA structures. The structure of Mgm101 bound with two complementary ssDNAs shows the second strand base-paired directly on top of the first, capturing an extended duplex intermediate of annealing. Our structures also reveal a novel positively charged beta-hairpin not present in RAD52, located at the outer rim of the Mgm101 ring. Native-MS and mass photometry confirm that the complexes observed in Cryo-EM snapshots are also present in solution, and indicate that Mgm101 also binds to pre-formed dsDNA. We hypothesize the novel beta-hairpin is somehow involved in dsDNA binding, and Cryo-EM structures of Mgm101 with pre-formed dsDNA are in progress. Altogether, our data suggest that Mgm101 anneals ssDNAs on the same ring in cis, and support a common mechanism for the SSAP superfamily from bacteriophage to humans.
Keywords: DNA repair, Cryo-EM, Native Mass Spectrometry