Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA

Klenow-DNA complex is known to undergo a rate-limiting, protein conformatio nal transition from an 'open' to 'closed' state, upon binding of the 'corre ct' dNTP at the active site. In the 'closed' state, Mg2+ mediates a rapid c hemical step involving nucleophilic displacement of pyrophosphate by the 3' hydroxyl of the primer terminus. The enzyme returns to the 'open' state up on the release of PPI and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the additi on of the next dNTP, without the preceding chemical step, we studied the te rnary complex (Klenow-DNA-dNTP) in the absence of Mg2+. While the ternary c omplex is proficient in chemical addition of dNTPs in Mg2+, as revealed by primer extensions, the same in Mg2+-deficient conditions lead to non-covale nt (physical) sequestration of first two 'correct' dNTPs in the ternary com plex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP-DNA complex is found to be stable even after the dissociation of Klenow. This reveals the novel state of the dNTP-DNA co mplex where the complementary base is stacked in a DNA-helix non-covalently , without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg2+-deficient ternary complex ex hibits a 'closed' conformation.