Interaction between DNA Polymerase lambda and anticancer nucleoside analogs

Abstract

The anticancer activity of cytarabine (AraC) and gemcitabine (dFdC) is thought to result from chain termination after incorporation into DNA. To investigate their incorporation into DNA at atomic level resolution, we present crystal structures of human DNA polymerase lambda (Pol lambda) bound to gapped DNA and containing either AraC or dFdC paired opposite template dG. These structures reveal that AraC and dFdC can bind within the nascent base pair binding pocket of Pol lambda. Although the conformation of the ribose of AraCTP is similar to that of normal dCTP, the conformation of dFdCTP is significantly different. Consistent with these structures, Pol lambda efficiently incorporates AraCTP but not dFdCTP. The data are consistent with the possibility that Pol lambda could modulate the cytotoxic effect of AraC.

FIGURE 1.

The structures of nucleoside analogs.

FIGURE 2.

Incorporation of the AraCTP and dFdCTP by Pol λ. Pol λ was incubated with a double-stranded DNA oligonucleotide containing a single nucleotide gap under the conditions described under “Materials and Methods” in the presence of different nucleoside triphosphates at 37 °C for 5 min. The reactions were analyzed using a denaturing sequencing gel, and the results were visualized with a phosphor imager. The reactions were control DNA without dNTPs (lane 1) and in the presence of dCTP (lane 2), AraCTP (lane 3), and dFdCTP (lane 4).

FIGURE 3.

Structure of Pol λ in complex with AraCTP and dFdCTP. A, the complex in which AraCTP is bound as an incoming nucleotide shows minimal distortion in the active site, and AraCMP has been fully incorporated into the primer chain. The AraCMP residue is overlaid with a normal dCTP residue (31). A simulated annealing F_o − F_c electron density map contoured at 3σ is shown (blue). B, structure of Pol λ in complex with dFdCTP. Although dFdCTP can be bound in the Pol λ active site, it can only be accommodated in a conformation that differs significantly from that of a natural dCTP (transparent). A simulated annealing F_o − F_c electron density map contoured at 3σ is shown (blue). C, two fluorine atoms have been modeled on the C2′ of a natural dCTP (31). The two fluorine atoms (Van der Waals surface in magenta) would clash with the backbone of active site residues and the side chain of Asn⁵¹³ (green). D, an overlay view of AraCTP and dFdCTP within the active center of Pol λ. The primer strand of the AraC structure is yellow. The primer strand for the dFdCTP structure is brown. The three catalytic aspartates are shown in magenta (AraC) and green (dFdCTP). E, incorporation of AraCTP and dFdCTP by Y505A Pol λ mutant protein. The wild type or Y505A mutant Pol λ was incubated with a double-stranded DNA oligonucleotide in the presence of different nucleoside triphosphates at 37 °C for 3 min under the conditions described under “Materials and Methods.”

FIGURE 4.

The inhibitory effects of dFdCTP on the incorporation of dCTP by Pol λ. The reaction conditions were the same as described under “Materials and Methods” except that increasing concentrations of dFdCTP (0, 0.5, 1, 5, and 20 μm) were added to the reactions of each dCTP concentration (0.1, 0.2, 0.5, 1, 1.5, and 2 μm).

FIGURE 5.

Cell sensitivity of mouse Pol λ^+/+ and Pol λ^−/− MEF cells to AraC. The SV40-transformed Pol λ^+/+ and Pol λ^−/− MEF cells were treated with increasing concentrations of AraC under the conditions described under “Materials and Methods.” The cell survival data are presented as percentage of the absorbance of treated/untreated cells. Error bars, S.D.