| The human genome is subject to alternations by both normal metabolic activities and environmental factors, resulting in many different types of DNA damage. Double-stranded DNA breaks (DSBs) are a particularly deleterious form of DNA damage. Mutations of the genes responsible for their repair can cause cancer and related diseases. In Chapter 1, an overview of the three pathways of DSB repair is given. One pathway for the repair of dsDNA breaks is single-strand annealing (SSA), which is promoted by Rad52 in eukaryotes, and by the phage-encoded RecET and Redalphabeta recombination systems in bacteria. RecET and Redalphabeta each consist of a 5'-3'exonuclease, RecE or Redalpha (lambda exonuclease), that resects the ends of the DNA created at the break to form long 3'-overhangs, and a second protein, RecT or Redbeta (beta protein), that loads onto the overhang to promote its annealing with a complementary strand of ssDNA. Due in large part to a lack of structural information, the proteins of these recombination systems are not well understood at the mechanistic level. The biotechnology applications of the phage-based recombination systems in gene manipulation and DNA sequencing are also introduced in this chapter.;In Chapter 2, the crystal structure of the C-terminal nuclease domain of RecE exonuclease at 2.8 A is presented. RecE forms a toroidal tetramer with a central tapered channel that is wide enough to bind dsDNA at one end, but is partially plugged at the other end by the C-terminal segment of the protein. Four narrow tunnels, one within each subunit of the tetramer, lead from the central channel to the four active sites, which lie about 15 A from the channel. The structure suggests a mechanism in which dsDNA enters though the open end of the central channel, the 5'-ended strand passes through a narrow tunnel to access one of the four active sites, and the 3'-ended strand passes through the plugged end of the channel at the back of the tetramer.;Based on the crystal structure and sequences comparisons, 24 amino acid residues of RecE were mutated to alanine are purified, and the nuclease and DNA-binding activities of the purified proteins are presented in Chapter 3. The structure-activity analysis, which targeted the six conserved active site motifs, the disordered loop that is positioned to interact with an incoming DNA substrate, the surface of the central channel, and the C-terminal plug, complements our structural study of RecE and supports the proposed model for its mechanism of action.;In order to expand our understanding of the molecular mechanism of this class of toroidal exonuclease enzymes, the crystallization of RecE and lambda exonuclease in complex with DNA was pursued and is still ongoing. In Chapter 4, we show that several forms of promising crystals of enzyme-DNA complexes have been obtained and the existence of DNA in the crystals was verified. We also discuss the strategies to facilitate the complex co-crystallization as a future direction, with particular emphasis on the design of DNA substrates with varied length and ends.;The Src homology 2 (SH2) domain specifically recognizes a phosphorylated tyrosine (pTyr) residue. This process is fundamental in many signal transduction events. In Chapter 5, we study the structural basis of the specificity for the interactions between SH2-domains and pTyr-containing sequences selected from the peptide library screenings, a collaborative project with Dr. Dehua Pei in Department of Chemistry at The Ohio State University. Crystal structures of N-terminal SH2 domain of SHP-2 phosphatase in complex with two different high affinity peptides, FVP and AQLW, were determined. A striking feature of the SH2-FVP structure is that two copies of the FVP peptide, running antiparallel to one another, bind to the peptide-binding surface on the SH2 domain. All previous structures contain only one peptide bound per SH2 domain. The biological implications of this novel 1:2 SH2 domain-peptide complex are discussed. |
