NIGEL D. F. GRINDLEY
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 very 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.
IS607 is a recently described transposable element from Helicobacter pylori that uses a serine recombinase as a transposase. In contrast to the vast majority of serine recombinases, the IS607 transposase catalyses intermolecular recombination (not only excising the element as a circle from a donor site but also reinserting it into new target sites) and it exhibits no obvious specificity for its target sites. The transposase also has its DNA binding domain at the N-terminus instead of at the conventional C-terminal position. We have initiated studies of the IS607 transposase with the aim of determining the features that account for its altered topological specificity and its remarkably relaxed site-specificity.
DNA Polymerases (for details see Catherine Joyce).
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.
Grindley, N.D.F., Whiteson, K.L. and Rice, P.A. Mechanisms of site-specific recombination. Ann. Rev. Biochem. 75, 567-605 (2006)
DeLucia, A.M., Chaudhuri, S., Potapova, O., Grindley, N.D.F. and Joyce, C.M. The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases. J. Biol. Chem. 281, 27286-27291 (2006)
Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D.F. and Steitz, T.A. Structure of a synaptic gamma-delta resolvase tetramer covalently linked to two cleaved DNAs. Science 309, 1210-1215 (2005)
Purohit, V., Grindley, N.D.F. and Joyce, C.M. (2003) Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment). Biochemistry 42, 10200-10211
Leschziner, A.E. and Grindley, N.D.F. The architecture of the gamma-delta resolvase crossover site synaptic complex revealed by using constrained DNA substrates. Molec. Cell 12, 775-781 (2003)