Poster abstracts

Poster number 21 submitted by Christopher Smith

Functional and structural characterization of Redβ: a unique single-strand annealing protein.

Christopher E. Smith (Ohio State Biochemistry Program)

Abstract:
Bacteriophage λ encodes a two-component synaptase-exonuclease (Syn-Exo) system used for generating end-to-end concatamers of λ genome before packaging. Redα (λ exo) is a processive 5’-3’ exonuclease that degrades linear dsDNA, yielding a 3’ ssDNA overhang. Redβ is a single-strand annealing protein (SSAP) that binds to the resulting 3’ overhang and anneals it to a complementary ssDNA. The current model for DNA binding and annealing describes Redβ binding relatively weakly to ssDNA as an oligomeric ring of 10-15 subunits, and forming a very tight complex in the form of a left-handed helical filament once the bound single-stand is annealed to a complementary ssDNA. Redβ serves as a model to study the unique DNA repair mechanism of single-strand annealing, which is conserved in higher organisms. Recently, phage-derived Syn-Exo partners have been used in restriction endonuclease- and ligation-independent genetic engineering, known as recombineering. We have identified a protease-resistant fragment of Redβ (residues 1-177), which is a target for structure determination via x-ray crystallography. We predict the N-terminal fragment forms the DNA binding domain, while a more flexible C-terminal tail modulates interaction with the partner exonuclease. While Redβ177 retains the ability assemble into oligomers, the functional capabilities of the two protein forms differ. Using a fluorescence-based assay, we characterized their DNA-binding properties and found RedβFL preferentially binds to sequentially-added complementary oligonucleotides, while Redβ177 binds more tightly to single-stranded oligonucleotides. Using a Ni-affinity pulldown assay, Red177 fails to interact with its intrinsic synaptosome partner, λ exonuclease. Further, utilizing an in vivo recombination assay, we found Redβ177 is unable to recombine a double-stranded PCR product or single-stranded oligonucleotide with a target plasmid containing regions of homology. Our results provide insight into how Redβ and other SSAP perform their DNA binding and pairing function in vivo.

Keywords: DNA Repair, Homologous recombination