Systematic biochemical analysis of somatic missense mutations in DNA polymerase β found in prostate cancer reveal alteration of enzymatic function

Abstract

DNA polymerase β is essential for short-patch base excision repair. We have previously identified 20 somatic pol β mutations in prostate tumors, many of them missense. In the current article we describe the effect of all of these somatic missense pol β mutations (p.K27N, p.E123K, p.E232K, p.P242R, p.E216K, p.M236L, and the triple mutant p.P261L/T292A/I298T) on the biochemical properties of the polymerase in vitro, following bacterial expression and purification of the respective enzymatic variants. We report that all missense somatic pol β mutations significantly affect enzyme function. Two of the pol β variants reduce catalytic efficiency, while the remaining five missense mutations alter the fidelity of DNA synthesis. Thus, we conclude that a significant proportion (9 out of 26; 35%) of prostate cancer patients have functionally important somatic mutations of pol β. Many of these missense mutations are clonal in the tumors, and/or are associated with loss of heterozygosity and microsatellite instability. These results suggest that interfering with normal polymerase β function may be a frequent mechanism of prostate tumor progression. Furthermore, the availability of detailed structural information for pol β allows understanding of the potential mechanistic effects of these mutants on polymerase function.

FIGURE 1

Identification of purified pol β protein and gel assay of pol β single nucleotide insertion. A: Electrophoretic analysis of the expressed pol β protein at various stages of purification. Separation was performed on a 12.5% (W/V) SDS-polyacrylamide gel. M, marker; Lane 1, crude extract from BL21 (DE3) E. coli cells containing pET-28a(+)/ pol β ; Lane 2, crude extract from IPTG-induced BL21 (DE3) E. coli cells containing pET-28a(+)/ pol β ; Lane 3, purified pol β . B: Detection of purified protein by western bolt. M, marker; Lane 1, purified wild type pol β. C: A gapped oligo substrate was incubated with wild type pol β in increasing concentrations of a single dNTP as described under “Experimental Procedures”. Pol β concentrations was adjusted to optimize detection of primer extension products. dATP misincorporation resulted in a single nucleotide extension of the misinserted dATP (correct extension against the next nucleotide (dT) of the DNA substrate) but only at higher dATP concentrations.

FIGURE 2

Influence of pol β variants on catalytic efficiency for dCTP insertion. WT and mutant variants were assayed on a single-nucleotide gapped DNA substrate with a templating dG, and the catalytic efficiency (k_cat/K_m,dNTP) was determined from dividing k_cat and K_m values obtained by Excel analysis of the inverse plots. These data represent the mean of at least three independent determinations, +/− standard error.

FIGURE 3

Relative fidelity of pol β enzyme variants. WT and mutant variants were assayed on a single-nucleotide gapped DNA substrate with a templating dG. Fidelity was calculated as described under “Experimental Procedures” and listed on Table 3. These data represent the mean of at least three independent determinations.

FIGURE 4

Location of the somatic pol β mutations. The structure of human DNA polymerase beta with dUTP (orange crosses) opposite dA and a gapped DNA substrate (2FMS) is shown using Jmol (http://molvis.sdsc.edu/fgij/fg.htm?mol=2FMS). Yellow “halos” indicate the side groups of the mutated residues. The gapped DNA substrate is shown in green and yellow, the polymerase β in gray. Small green spheres indicate Mg²⁺ ions, large green ones Cl⁻ ions, and purple ones Na⁺ ions.