Berlin 2012 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 13: DNA/RNA and Related Enzymes

BP 13.10: Vortrag

Mittwoch, 28. März 2012, 12:15–12:30, H 1058

The partially closed conformation of DNA polymerase I provides a decision point for nucleotide selection — •Johannes Hohlbein¹, Catherine Joyce², and Achillefs Kapanidis¹ — ¹Biological Physics Research Group, Dept. of Physics, University of Oxford, UK — ²Dept. of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, U.S.A.

The high fidelity of many DNA polymerases depends largely on conformational changes that precede the chemical step of phosphoryl transfer and serve as checkpoints to reject inappropriate substrates early in the reaction. One of these conformational changes is the fingers-closing transition, during which the fingers subdomain moves from an open to a closed conformation.

Here, we use single-molecule FRET to resolve conformational changes within the bacterial DNA polymerase I with sub-nanometre resolution. We compared the wild-type polymerase to derivatives bearing single amino-acid substitutions at residues E710 and Y766, both which are invariant within the A family of DNA polymerases.

Our results show that these derivatives have decreased affinity for the complementary dNTP, and do not perform efficient fingers-closing. Instead, intermediate FRET states are populated, which are likely to correspond to a fidelity-associated partially closed state of the fingers.

These differences in the interactions and conformations formed along the reaction path reduce discrimination between complementary and non-complementary nucleotides, and provide a basis for the reduced fidelity of the derivatives.