Novel Conformational States in Mutator DNA Polymerases Observed Using Single-Molecule FRET

Abstract

The remarkable fidelity of DNA polymerases depends largely on their efficient rejection of incorrect nucleotides prior to nucleotide addition. Previously, we used single-molecule FRET to examine fidelity-related conformational transitions preceding nucleotide addition by DNA polymerase I (Klenow fragment). Our experiments distinguished the open and closed conformations that predominate in the binary Pol-DNA and ternary Pol-DNA-dNTP(complementary) complexes, and showed that the unliganded polymerase is highly conformationally flexible. We also showed that ternary complexes with mismatched dNTPs or complementary ribonucleotides form novel FRET species perhaps corresponding to partially closed conformations which may act as kinetic checkpoints crucial for fidelity.Here, we studied how amino acids proximal to the polymerase active site contribute to fidelity by examining the conformational states of polymerase derivatives that act as “mutators”, i.e., have decreased fidelity. For example, an E710A substitution (glutamic acid to alanine) reduces the fidelity through a mechanism that remains unclear. Our studies of a doubly labelled E710A polymerase (Pol[E710A]) showed that the binary complex favors the open conformation more than the wild-type. More intriguingly, addition of the complementary dNTP to a binary Pol[E710A]-DNA complex does not form the closed conformation seen in the ternary complex of the wild-type polymerase; in contrast, and at high nucleotide concentrations, Pol[E710A] adopts a FRET state similar to that in ternary complexes of wild-type polymerase with mismatched dNTPs or complementary ribonucleotides; this state may correspond to a partially closed conformation, and is also adopted by Pol[E710A]-DNA-dNTP(mismatched) complexes, offering a possible explanation for the reduced fidelity associated with E710A. Since Pol[E710A] can perform DNA synthesis, our results raise the question whether polymerisation occurs from a partially closed conformation or a transiently formed closed complex; experiments to address this question are in progress, along with studies of additional mutators.