Promiscuous mismatch extension by human DNA polymerase lambda

Abstract

DNA polymerase lambda (Pol lambda) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol lambda to extend mismatched primer-termini, either on 'open' template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5'-phosphate. Interestingly, Pol lambda extended mismatches with an average efficiency of approximately 10(-2) relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were approximately 260-fold higher than on template-primer molecules. A crystal structure of Pol lambda in complex with a single-nucleotide gap containing a dG.dGMP mismatch at the primer-terminus (2.40 A) suggests that, at least for certain mispairs, Pol lambda is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol lambda suggests a potential role as a 'mismatch extender' during non-homologous end joining (NHEJ), and possibly during translesion synthesis.

Figure 1

Human Pol λ directly extends a mismatched end. Two different template-primer structures were used, differing in the 3′ end nucleotide of the primer. Extension of the 5′ end labeled primer (^*) was examined by PAGE. Reactions were carried out as described in Materials and Methods.

Figure 2

3D-structure of the human Pol λ core complexed with a 1 nt gapped-DNA having a dG·dGMP mismatched primer-terminus. (A) View of the Pol λ active site showing the dG·dGMP mismatch (yellow). The template and primer strands are shown in gray and the protein is shown in green. A simulated annealing Fobs-Fcalc omit electron density map where the terminal base pair was omitted, contoured at 2.5 σ, is shown (blue). (B) Bonding distances between the template base (dG) and the 3′ primer nucleotide (dG) forming a dG·dGMP mismatch corresponding to the structure shown in (A). Hydrogen bonds are shown as dotted lines. Water molecule is represented as a purple ball. (C) Stereo-view showing the superimposition of the dG·dGMP terminal mismatch (colored) and a correct dG·dCMP base pair (gray) derived from a binary complex of Pol λ (PDB entry 1XSL). (D) Stereo-view showing the superimposition of the dG·dGMP terminal mismatch (colored) and a correct dA·dTMP base pair (gray) derived from the ternary complex of Pol λ (PDB entry 1XSN).

Figure 3

Mismatch extension efficiencies of different DNA polymerases. Mismatch extension efficiencies (f_ext°) of different DNA polymerases: human Pol κ [dark blue; (40)], yeast Pol ζ [green; (41)], human Pol λ (red; obtained from Table 3), human Pol β [yellow; (48)] and Drosophila melanogaster Pol α [light blue; (37)].