Bill Konigsberg, PhD

Molecular Biology and Structure of a DNA Replicase

The current objective of our laboratory is to determine the mechanisms used by B family DNA polymerases for base discrimination. We have selected a T even phage DNA polymerase, RB69 pol, as a prototype for several reasons: (i) it has considerable sequence similarities to human DNA pol a and d; (ii) it is assembled into a DNA replicase with accessory proteins in an analogous fashion to human DNA replicases; (iii) there is a wealth of structural and kinetic information about this enzyme that enables new findings about the mechanism of action of RB69 pol to be interpreted within a well established framework; (iv) despite extensive studies it is still unclear how RB69 as well as other DNA pols are able to replicate DNA with a minimum of errors (2x10-8 per base per genome replication). To understand how this level of fidelity is achieved we are investigating the kinetic behavior of the wild type and selected RB69 polymerase mutants complexed with short dsDNA fragments (P/T) when challenged with correct or with non-complementary dNTPs. Among the wide variety of experimental approaches that we are using to investigate the mechanisms employed by RB69 pol for faithful DNA replication are: (i) rapid chemical quench to estimate pre-steady-state kinetic parameters for wild type and RB69 pol mutants with amino acid substitutions that affect fidelity; (ii) stopped-flow fluorescence to determine rates of conformational changes during the polymerization and editing cycles; (iii) single molecule fluorescence and single molecule FRET to monitor the dynamics of the nucleotidyl transfer reaction and to detect conformationally distinct transient intermediates that may have a profound influence on fidelity. (iv) x-ray crystallography to determine the structure of RB69 pol mutants that display dramatically reduced base discrimination while maintaining pre-steady-state kinetic parameters for incorporation of correct dNMPs that match those of the wild type enzyme. From the results of these experiments we hope to be able to identify the rate limiting step for the incorporation of correct and incorrect bases.

Ultimately we hope to assemble the complete DNA replication complex, and elucidate the mechanisms that are responsible for the faithful replication of the T even phage genome. We believe that detailed information about this system will be valuable in deciphering the enzymology of viral and mammalian replication systems.