14] Solution structure of DNA polymerases and DNA polymerase-substrate complexes

Abstract

For the solution characterization of structure in DNA polymerases multidimensional nuclear magnetic resonance spectroscopy (NMR), nuclear overhauser effect (NOE) studies, water proton relaxation rate (PRR) measurements, and electron paramagnetic resonance spectroscopy (EPR) are used. Studies included structure determination of a domain of DNA polymerase β (Pol β), determination of the conformations of substrates bound to DNA polymerase I, and the determination of the location and affinity of divalent metal binding sites on DNA polymerase I. NMR and EPR methods provided the first structural insight into the mechanism of Escherichia coli DNA polymerase 1. Studies of the cloned Klenow fragment provided information on substrate binding sites, the interaction between substrate and metal binding sites, and substrate conformation. Methods of 2D and 3D heteronuclear NMR are applicable to the determination of solution structures of domains of DNA polymerases with molecular weights of less than 30,000. High-resolution structural analysis is presently being directed toward the single-stranded template-binding domain of the enzyme and the study of the interaction of this domain with single-stranded DNA. The chapter discusses most recent NMR methods that are being used to study Pol β, together with of an overview of previous methods used in the study of DNA polymerase I.