A unifying theme of our research is the study of enzymes that make and break phosphodiester bonds in DNA. Our research focuses on the detailed biochemical mechanisms of (i) site-specific recombination mediated by the prototypical serine recombinase, gd resolvase, and (ii) DNA synthesis and degradation mediated by the DNA polymerases Pol I (of E. coli), a highly accurate family A polymerase, and Dbh, an inaccurate family Y polymerase, which acts specifically to bypass damaged bases in the template strand during replication. In all three cases, we have detailed structural information obtained through collaborations with the X-ray crystallography group of Tom Steitz. In addition to using standard biochemical methods, we have recently added fluorescence techniques to dissect the biochemical pathways and define the nature and the role of the conformational transitions that take place during the processes of recombination or polymerase action.
Extensive Research Description
Mechanisms of Protein-DNA transactions.
Our research group is studying the mechanisms of a variety of enzymes that make, break, or rearrange DNA. Our work involves a mixture of biochemistry and genetics, and in several instances is strongly influenced by very successful collaborations with the structure group of Tom Steitz.
Serine recombinases and site specific recombination.
Gamma-delta resolvase is the prototype of a large family of site-specific recombinases that use a specific serine residue as the nucleophile for cutting and rejoining defined DNA segments. The serine recombinases make concerted double strand breaks in the two recombination sites before any exchange and resealing of DNA strands occurs. Phosphodiester bond energy is conserved by formation of a covalent resolvase-DNA (phospho-serine) linkage to the 5' ends of the transiently broken DNA strands. Gamma-delta resolvase performs site-specific recombination in an elaborate synaptic complex containing 12 resolvase subunits and two 114 base pair DNA segments (called res) each with three specific dimer binding sites. We recently proposed a new model for the synaptic complex, using a combination of structural information and a detailed analysis of the various interactions between resolvase protomers that are responsible for the assembly and function of the active complex. A strong implication of the model is that the two crossover sites are on the outside of the complex, well separated from one another. This feature has been demonstrated both by biochemical studies and by a recent crystal structure of a simplified resolvase synaptic complex (four subunits with cleaved crossover sites) solved in the Steitz lab. Current goals include testing implications of this synaptic structure for strand exchange, and determining how this structure fits into the full (12 subunit) synaptic complex.
Our goal is a structural and mechanistic understanding of the reactions involved in DNA replication, using simple DNA polymerases of known three-dimensional structure as model systems. Currently, we are exploring the basis of polymerase accuracy in two contrasting polymerases: the highly accurate DNA polymerase I of E. coli, and the very inaccurate Dbh lesion bypass polymerase. We are also using fluorescence techniques to define the nature and the role of the conformational transitions that take place during the polymerase reaction.
- Bai, H., Sun, M., Ghosh, P., Hatfull, G.F., Grindley, N.D.F. and Marko, J.F. (2011) Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase Proc. Nat. Acad. Sci. USA; published ahead of print April 1
- Bermek, O., Grindley, N.D.F. and Joyce, C.M. (2011) Distinct roles of the active site Mg2+ ligands, D882 and D705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions. J. Biol. Chem. 286, 3755-66 [E. pub. Nov. 16, 201
- Santoso, Y., Joyce, C.M., Potapova, O., Le Reste, L.,Hohlbein, J., Torella, J.P., Grindley, N.D.F. and Kapanidis, A.N. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. Proc. Nat. Acad. Sci. USA 107, 715-20
- Joyce CM, Potapova O, Delucia AM, Huang X, Basu VP, Grindley ND. Biochemistry. 2008 Jun 10;47(23):6103-16. Epub 2008 May 13.
- Grindley, N.D.F., Whiteson, K.L., and Rice, P.A. (2006). Mechanisms of site-specific recombination. Ann. Rev. Biochem. 75:567-605.
- Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D.F., and Steitz, T.A. (2005). Structure of a synaptic ƒÁƒÂ resolvase tetramer covalently linked to two cleaved DNAs. Science 309:1210-1215.