Interdisciplinary Graduate Programs Symposium
2014 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
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Poster abstracts
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 digests 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 Redβ DNA binding and annealing describes β binding weakly to ssDNA as an oligomeric ring of 10-15 subunits, and forming a very tight complex with nascent annealed duplex in the form of a left-handed helical filament. Redβ serves as a model to study the unique DNA repair mechanism of single-strand annealing, which is conserved in higher organisms and includes the eukaryotic homologous pairing protein Rad52. 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 that the N-terminal fragment forms the DNA binding domain, while a more flexible C-terminal tail modulates interaction with the partner exonuclease. Full-length Redβ and Redβ(177) are able to assemble into oligomeric structures, but their functional properties differ significantly. Using a fluorescence-based assay, we characterized the DNA-binding and annealing properties of Redβ(177) compared to that of full-length protein and found Redβ(FL) preferentially binds to sequentially-added complementary oligonucleotides, while Redβ177 binds more tightly to ss oligonucleotides. Further, using a Ni-affinity pulldown assay, Redβ(177) fails to interact with λ exonuclease. Utilizing an in vivo assay, we found Redβ(177) is unable to recombine a PCR product or ss oligo with a target plasmid containing regions of homology. Our results provide insight into how SSAP perform their DNA binding and pairing function in vivo.
References:
1. Passy SI, Yu X, Li Z, Chiu SK, Reddy G, and Radding CM (1999). Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc Natl Acad Sci USA 96: 4279-4284.
2. Poteete AR (2001). What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS Microbiol Lett. 201, 9-14.
3. Wu Z, Xing X, Wisler JW, Dalton JT, and Bell CE (2006). Domain structure and DNA binding regions of β protein from bacteriophage λ. J Biol Chem 281, 25205-25214.
Keywords: Single-strand annealing, Recombination, DNA Repair
