Nigel Grindley, PhD

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.

DNA Polymerases
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.