Structural basis for base discrimination by RB69 DNA polymerase

Abstract

Replicative DNA polymerases (pol) copy genomes efficiently with high base selectivity. Kinetic studies have shown that in the RB69 pol triple mutant, L561A/S565G/Y567A, in which residues in the nucleotide binding pocket that contact the nascent base pairs, were reduced in size, base selectivity drops dramatically but the kinetic parameters for incorporation of correct bases was unaltered relative to the wild type enzyme. To provide the structural basis for this kinetic discrimination, we solved crystal structures of the RB69 pol triple mutant in the presence and absence of dA‐dTTP and in complex with mismatched base pairs. The apo form of the mutant RB69 pol was captured in a unique, half‐closed state. Helix P in the apo form of the pol mutant was straight, eliminating the necessity of the kinked‐to‐straight conversion that occurs in wild type pol when it forms the ternary complex. Also, the phenyl ring of Y567, at the kink of helix P, which contacts the templating base, rotates 25° during conversion to the ternary complex. The extent of this rotation is limited by its interaction with Y416, Y391 and I586. This constraint is eliminated in the triple mutant and, as a consequence, base selectivity is reduced. L561 and S565 also contact the templating base, restricting its flexibility. When these two residues are replaced in the triple mutant, base discrimination is further reduced.