Structural Biology of DNA Repair by Single-Strand Annealing

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Intellectual Merit:The cells of all organisms are exposed constantly to environmental insults that damage their DNA and the genetic information it encodes. Double stranded DNA (dsDNA) breaks, in which both strands of the duplex are broken at the same position, are particularly harmful. Fortunately, cells have many ways of repairing dsDNA breaks. An important repair pathway, called "single-strand annealing" (SSA), involves resection (trimming back) of the DNA ends exposed at the break to form two single-stranded DNA overhangs, which are then annealed to one another to repair the break. SSA is promoted by a network of proteins including Rad52 in eukaryotes, or by the relatively simple phage-encoded RecET and Redab (Red "alpha/beta") recombination systems in bacteria. The RecET and Redab recombination systems each consist of two proteins: a highly processive 5'-3' exonuclease, RecE or Reda, which binds to dsDNA ends and digests the 5'-ended strand, and a single-strand annealing protein, RecT or Redb, which binds to the resulting 3'-overhang to promote its annealing with a complementary strand of single-stranded DNA (ssDNA). Interestingly, the two proteins of each system bind to one another to form a complex known as a "synaptasome," which may serve to load the single-strand annealing protein onto the 3'-overhang as it is generated by the exonuclease. The RecET and Redab recombination systems are highly evolved and efficient, and offer a convenient model for understanding the basic mechanistic principles of SSA. Moreover, due to their ability to work at short regions of homology, RecET and Redab have recently been deployed to create powerful new methods for genetic engineering called "recombineering." The exonuclease enzymes are also being exploited in new methods for single-molecule nanopore DNA sequencing. In spite of the importance of RecET and Redab as model systems, and their emergence in powerful new biotechnology applications, the proteins are not well understood at the mechanistic level, in large part due to a lack of structural information. The long-term goals of this project are to apply the tools of structural biology, biochemistry and genetics to elucidate the inner workings of the RecET and Redab recombination systems at the atomic level. In Aim 1, x-ray crystal structures of RecE and Reda in complex with DNA substrates will be determined, to reveal how they bind to dsDNA ends and processively digest DNA substrates. In Aim 2, crystal structures of RecT and Redb will be determined, to reveal how they bind to ssDNA and promote the annealing of complementary strands. These studies will provide a foundation for understanding the underlying mechanistic principles of SSA proteins. The knowledge gained from these studies will also pave the way for the design of new proteins with enhanced properties for applications in genetic engineering and nanopore DNA sequencing.Broader ImpactsThis project will provide rich opportunities for the training of graduate, undergraduate, and high school students. Graduate students will be recruited from established programs at OSU, including the Ohio State Biochemistry Program (OSBP), the Biophysics Graduate Program, and the Chemistry-Biology Interface Program. The PI will also provide research and training opportunities to undergraduate students from under-represented minority backgrounds, recruiting through OSU's Summer Research Opportunities Program (SROP). In addition, the PI has a partnership with Metro High School in Columbus, Ohio to recruit high school students for paid summer internships. Metro is a newly formed, STEM-focused high school near OSU that gives students from low-income, urban neighborhoods the opportunity to participate in an advanced, early college, science-based curriculum. Metro students typically begin taking classes at OSU during their Junior years, and are encouraged to participate in hands-on research activities that reinforce their classroom studies. Towards this end, the PI will recruit one Metro student each year to work in the laboratory as a paid summer intern. The students will be given the opportunity to participate in all aspects of x-ray structure determination, including cloning, expression, and crystallization of target proteins, as well as x-ray structure determination and analysis. The PI will also participate in activities at Metro High School through his service on the Biomedical Partnership Team, a committee of local scientists that helps to design innovative science curricula for advanced area high school students.