Y265C DNA polymerase beta knockin mice survive past birth and accumulate base excision repair intermediate substrates

Abstract

DNA is susceptible to damage by a wide variety of chemical agents that are generated either as byproducts of cellular metabolism or exposure to man-made and harmful environments. Therefore, to maintain genomic integrity, having reliable DNA repair systems is important. DNA polymerase β is known to be a key player in the base excision repair pathway, and mice devoid of DNA polymerase beta do not live beyond a few hours after birth. In this study, we characterized mice harboring an impaired pol β variant. This Y265C pol β variant exhibits slow DNA polymerase activity but WT lyase activity and has been shown to be a mutator polymerase. Mice expressing Y265C pol β are born at normal Mendelian ratios. However, they are small, and 60% die within a few hours after birth. Slow proliferation and significantly increased levels of cell death are observed in many organs of the E14 homozygous embryos compared with WT littermates. Mouse embryo fibroblasts prepared from the Y265C pol β embryos proliferate at a rate slower than WT cells and exhibit a gap-filling deficiency during base excision repair. As a result of this, chromosomal aberrations and single- and double-strand breaks are present at significantly higher levels in the homozygous mutant versus WT mouse embryo fibroblasts. This is study in mice is unique in that two enzymatic activities of pol β have been separated; the data clearly demonstrate that the DNA polymerase activity of pol β is essential for survival and genome stability.

Fig. 1.

Pol B^Y265C/Y265C (c/c) mice are small. (A) Pol B^C/C embryos were found to be 33% ± 2% smaller than WT littermates at embryo day E11 and E14. No other noticeable physiological abnormalities were evident in any of the embryos. (B) The average body weight of the Pol B^c/c 1-d-old pups is approximately 1 g (Inset), whereas the Pol B^+/+ and Pol B^c/+ littermates were ∼1.4 g. The average weights of mice between days 1 and 21 shows that the Pol B^C/C mice gain weight at a slower rate compared with their Pol B^+/+ and Pol B^c/+ littermates. Data represent the mean weight of five to eight mice, with error bars showing the SEM.

Fig. 2.

Organs from Pol B^c/c embryos exhibit slow proliferation and high levels of cell death. (A) Immunohistochemistry results using the Ki67 marker showed cells in c/c embryos have fewer proliferative cells than WT littermates. Percentages are calculated by counting number of positively stained cells over the total number of cells using 10–15 images (×40) of immunohistochemistry sections representative of the same organ. (B) Immunohistochemistry sections of E14 embryos from various organs of homozygotes show high levels of apoptosis (±STD) in represented tissues. (Inset) Immunohistochemistry sections (40×) representative of brain and fetal liver with darker cells indicating the TUNEL-positive labeled cells. The homozygous mutant brains and fetal liver had 40-fold and sevenfold, respectively, increases in TUNEL-positive cells compared with WT. Represented P values are calculated using the unpaired t test.

Fig. 3.

Pol B^c/c MEFs exhibit decreased proliferation. (A) Homozygous mutant c/c MEFs were found to grow more slowly compared with WT and heterozygous littermates. Approximately 1.5 × 10⁵ cells from passage 1–2 MEFs were plated and then counted every other day. (B) Pol B^c/c MEFs accumulate in G2/M. Cell cycle analysis was performed by flow cytometry using propidium iodide (PI) to stain the DNA. Graph shows the numbers of cells in various cell cycle phases versus fluorescence intensity. (C) Increased level of phosphorylated ChK1 in c/c versus WT MEFs. (Upper) Images of Western blot; (Lower) quantification of the results. Lane 1, ^+/+ MEFs; lanes 2, C/C MEFs.

Fig. 4.

Increased levels of chromosomal aberrations of c/c primary MEFs. Diverse types of irregular Giemsa-stained chromosomes were counted from the metaphase spreads of more than 100 cells of c/c, c/+, and WT MEFs. As shown, homozygous c/c MEFs contain significantly (P < 0.05) elevated numbers of chromosomal aberrations compared with WT and c/+ MEFs. GraphPad prism. Two-way ANOVA was used to determine the P value. (Inset) Representative metaphase spreads from Pol B^c/c MEFs with arrows indicating two chromosomal irregularities: a chromosome fragment and a single chromatid break. N, number of metaphase spreads examined for each genotype.

Fig. 5.

The Pol B^c/c MEFs show sensitivity to MMS and are BER deficient. (A) Pol B^c/c MEFs are sensitive to MMS. Approximately 1.5 × 10⁵ cells from passage 1–2 MEFs were plated. On the following day, the cells were treated for 1 h with various concentrations of MMS and counted 5 d later. No differences were exhibited to treatment with UV light (Fig. S3). Data represent the mean of three independent experiments with error bars showing SEM. (B and C) Base excision repair assays of MEF cell extracts using either a Uracil (B) or hypoxanthine (C) substrate as described previously (12). BER assays were carried out as described in Experimental Procedures. (Upper) Images of the denaturing gel; (Lower) quantification of the results. Lane 1, annealed oligo substrate; lanes 2 and 3, substrate incubated with ^+/+ extract and in absence and in presence of dNTPs, respectively; lanes 4 and 5, substrate incubated with c/+ extract in absence and presence of dNTPs, respectively; lanes 6 and 7, substrate incubated with c/c extract and in absence and presence of dNTPs, respectively. (D) Western blot analysis shows expression of DNA polymerase β protein in WT (^+/+) and homozygous (c/c) mutant MEF cell extracts. (E) Y265C has dRP lyase activity. The 5′-dRP–containing DNA substrate (5 nM) was incubated in buffer R at 37 °C for 20 min with 100 nM of polymerase β (WT or Y265C), followed by the addition of 340 mM NaBH4 and stabilization of the reaction product as indicated in Experimental Procedures. Products were analyzed by a denaturing 20% polyacryamide gel and visualized by autoradiography.

Fig. 6.

Pol B^c/c MEFs accumulate intermediate BER substrates. (A) Pol B^c/c MEFs show increased levels of SSB. DNA tail moment of 100–125 cells of each genotype was quantified using CometScore software (Tritek). Pol B^c/c MEFs have significantly higher levels of SSBs than WT upon 1 and 2 h of exposure to 1.5 mM MMS. Represented P values are calculated using the unpaired t test. (B) Induced levels of γH2AX foci in the Pol B^c/c MEFs treated with MMS. Results are the average numbers of foci in 30–50 cells for each time point. Star and arrow show the time point where the MMS was removed (after 60 min exposure) and the cells were washed and allowed to recover for the time denoted. P values are calculated using GraphPad prism, unpaired t test. (C) Treated Pol B^c/c MEFs show increased levels of γH2AX compared with WT. (Upper) Images of a Western blot; (Lower) Quantification of the results. Lane 1, untreated ^+/+ MEFs; lanes 2 and 3, ^+/+ MEFs treated with 1.5 mM MMS for 1 h and recovered for 3 and 7 h, respectively; lane 4, untreated C/C MEFs; lanes 5 and 6, C/C MEFs treated with 1.5 mM MMS for 1 h and recovered for 3 and 7 h, respectively. (D) Pol B^c/c MEFs show significantly increased levels of γH2AX foci compared with WT.

Fig. 7.

Induced apoptosis in the Pol B^c/c MEFs exposed to methyl methanesulfonate (MMS). (A) Fold change of early and late apoptotic cells after MMS treatment with 1.5 mM MMS for 1 h followed by recovery for the represented time. Relative counts of Annexin V + 7-AAD–positive cells were measured using flow cytometry. (B and C). Effects of MMS treatment on early (PE Annexin V staining; lower right) and apoptosis (PE Annexin V and 7-AAD staining; upper right) was measured by flow cytometry. (B) FACs analysis of Pol B^+/+ MEFs after 24 h recovery following 1 h treatment with 1.5 mM MMS. (C) FACs analysis of Pol B^c/c MEFs after 24 h following 1 h treatment with 1.5 mM MMS. Representative examples of two to three separate experiments are shown.