Poster abstracts
Poster number 10 submitted by Brian Caldwell
Structure and mechanism of a bacteriophage Rad52 homolog in complex with a duplex intermediate of annealing.
Brian Caldwell ( Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology), Charles Bell ( Ohio State Biochemistry Program, Department of Biological Chemistry and Pharmacology)
Abstract:
Rad52 is a protein in humans that binds to ssDNA and promotes the annealing of complementary strands. This activity is involved in multiple DNA repair pathways, and mutants of Rad52 are synthetically lethal with mutations in BRCA1 and BRCA2, making it an active target for cancer therapeutics. Structures of the Rad52 DNA binding domain reveal an 11-mer ring that binds to ssDNA in an extended conformation with the bases exposed for homology recognition. However, there is currently no structure of Rad52 with two strands of DNA bound simultaneously, and its mechanism of annealing remains poorly understood. To approach this problem, we have turned to the RecT/Redβ family of single strand annealing proteins from bacteriophage, which are distant homologs of Rad52 that form stable complexes with a duplex intermediate of annealing. The best studied of these, Redβ from bacteriophage λ, forms oligomeric rings like Rad52 when bound to ssDNA substrate, but left-handed helical filaments when bound to an annealed duplex intermediate. These prior low-resolution structures led to a model for DNA annealing in the Rad52 family of proteins where the oligomeric rings bind to ssDNA, then transition (or reassemble) into a helical filament upon pairing with a complementary sequence from another DNA strand. Here, we have determined a 3.4 Å structure of a of a RecT homolog from a prophage of Listeria innocua. This is the first structure a Rad52 family homolog to be determined bound to two complementary strands of DNA simultaneously, and thus provides new insights into the possible mechanisms of annealing.
Keywords: CryoEM, Single Strand Annealing