Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase lambda

Abstract

The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase lambda bound to a primer-template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.

Figure 1

Crystal structures of the R517A polymerase λ mutant. (A) Overlay of molecule A (solid colour; see text) with a polymerase (pol) λ ternary complex (Protein Data Bank entry 1XSN; transparent). In the mutant binary complex, the template strand is located in an analogous conformation to that observed in a wild-type ternary complex, whereas Tyr 505 and Phe 506 are in a binary conformation. (B) In molecule B, two conformations are observed for the template strand, corresponding to the binary (yellow) and ternary (magenta) conformations. Two simulated annealing F_o−F_c omit density maps are shown, contoured at 3σ. In one case, the ternary-like conformation was omitted from map calculation (magenta), whereas in the other case the binary-like conformation was omitted (yellow). (C) The primer strand in molecule B is maintained in the same conformation by several protein–DNA interactions (see text), whereas the template strand adopts two alternative conformations.

Figure 2

Crystal structure of R517K polymerase λ. In both molecules in the asymmetric unit, a similar conformation of the Lys 517 side chain (cyan) is observed in a position to establish a water-mediated hydrogen bond with the base of the primer-terminal template nucleotide. A simulated annealing F_o−F_c omit density map is shown in blue, contoured at 6σ. Tyr 505 and Phe 506 are in positions intermediate between those observed in the binary and the ternary structures. The conformation of these side chains in the ternary complex is shown in grey.

Figure 3

Range of DNA motion during molecular dynamics simulations of wild-type and mutant (R517A and R517K) polymerase λ systems. The different panels show selected positions of the DNA backbone obtained from simulations of (A) wild-type polymerase λ, (B) R517A mutant, (C) R517K mutant and (D) R517K mutant with the catalytic ion. Arrows indicate ranges of DNA motion. The simulated DNA is superimposed on the crystal structures of the binary and ternary complexes (Protein Data Bank (PDB) entry 1XSL, green, and PDB entry 1XSN, red, respectively) to provide a frame of reference. The dTTP and Mg²⁺ from the ternary crystal structure are also shown in each panel. cat, catalytic ion bound; dTTP, 2′-deoxythymidine 5′-triphosphate; Nuc, nucleotide-binding ion bound; WT, wild-type polymerase λ.