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 Impacts
This 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.
Proteins of viral "SynExo" DNA recombination systems were studied, to understand the mechanism by which they achieve their functions. SynExo recombination systems use two proteins, a synaptase (Syn) and an exonuclease (Exo) to link multiple copies of the viral genome to one another, through a mechanism called single strand annealing. This helps the virus to replicate and package its DNA to make new viral particles. SynExo recombination systems are currently being exploited in powerful new methods for genome engineering. A better understanding of the inner workings of the proteins will facilitate their rational redesign for new and improved applications in biotechnology. The information gained will also be used to design antiviral drug compounds. Here is how the SynExo system works. First, the Exo protein binds to the end of the viral DNA, and begins to cleave one of the strands (the 5’-strand) into mononucleotides. This generates a 3’-overhanging single-stranded DNA (ssDNA), to which the second protein (Syn) binds as a large oligomeric ring. The Syn protein binds the ssDNA in such a way that its bases are exposed for homology recognition, so that it can be paired with a complementary strand from another DNA molecule. The net result is that two copies of the viral genome are spliced together to form an end-to-end concatemer for genome packaging. An important experimental tool used in the project was x-ray crystallography. This method allows us to essentially take pictures of the tiny protein molecules, by solving their three-dimensional structures at atomic (~1 Å, or a ten-millionth of a meter!) resolution. Having a high-resolution photograph, or 3D "structure" of a protein molecule tells us volumes about how the protein functions. The structure also provides a framework for mapping functional data onto the protein, such as how mutations at specific amino acid residues of the protein alter its functional properties. A big hurdle in getting the structure is that the protein molecule (or protein-DNA complex as was the case in this study) must be highly purified and coaxed to form highly ordered crystals, which are then exposed to the x-ray beam for the imaging process. Much of our work in the lab involves purifying the proteins and trying to grow crystals. One of the main outcomes of this study was that we solved the structure of the Exo protein in complex with its DNA substrate. The structure of the Exo protein had been determined previously, revealing that it forms a trimer (a complex of three identical copies of the protein) that is shaped like a doughnut. Based on this structure it had been hypothesized that the DNA binds to the central hole on the trimer, but the structure with DNA had not yet been determined. In order to obtain the structure of Exo bound to DNA, it was necessary to use a mutant form of the protein that binds to the DNA in the normal way, but does not cleave it, so that a "snapshot" of the protein frozen in action could be taken. This was achieved by mutating a critical active site lysine residue of the protein into alanine. The structure revealed that the DNA does bind to the hole on the doughnut, but is tilted so that one end of the DNA fits into the active site of one of the three subunits of the trimer (see Figure 1). Remarkably, the structure showed that enzyme unwinds the DNA, by two base pairs prior to cleavage, such that the 5’-strand feeds into one of the three active sites (Figure 2), while the 3’-strand threads through the central hole on the trimer. Based on this structure and on analysis of several targeted mutations in the protein, we have put forward a model for how the Exo protein moves along the DNA cleaving one nucleotide unit at a time. We also carried out experiments to try to crystallize the Syn protein, the one that binds to the overhaging ssDNA to anneal it to a complementary strand, both alone and in complex with DNA. We have not yet obtained crystals that are suitable to carry out the x-ray analysis, but significant progress has been made. Efforts to improve the crystals and see the structure of the Syn protein are currently ongoing. In addition to these two major scientific outcomes, the project has provided valuable training for two graduate students, two undergraduate students, and several (~10) high school students (Figure 3). A unique aspect of our project is that it provides an opportunity for advanced students from a local STEM-focused high school called Metro to gain hands-on experience in the research lab. The high school students work directly on the protein crystallization and x-ray diffraction experiments. Two high school students have even been authors on a peer-reviewed journal article in a top scientific journal.
