ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination

Abstract

Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1(-/-) pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1's potential involvement in DNA repair, fibroblasts were isolated from Alkbh1(-/-) mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1(-/-) and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1's role in class switch recombination, splenic B cells were isolated from Alkbh1(-/-) and Alkbh1(+/+) mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.

Figure 1. Scheme of ALKBH1’s AP lyase activity.

Figure 2. Characterization of ALKBH1 deficient mice.

A. Offspring distribution of crosses between heterozygous male and female mice. Numbers of pups from nine different litters are shown, demonstrating non-Mendelian inheritance of the knockout allele. B. Offspring distribution after crosses between Alkbh1^+/− females and Alkbh1^−/− males. C. Male-to-female ratios of Alkbh1^+/+, Alkbh1^+/−, and Alkbh1^−/− mice show a distorted sex ratio in favor of males for the knockout mice. D. Different sizes of Alkbh1^−/− and wild type (larger pup) littermates at two weeks of age. E. Weight development of Alkbh1^−/− and wild type pups. Alkbh1^−/− and wild type pups were derived from breeding either two ALKBH1 deficient or two wild type parent animals. For each time point, the weights of at least twelve animals were used to calculate the average and the error bars represent standard deviations.

Figure 3. ALKBH1 deficient fibroblasts are not more sensitive to DNA modifying agents than hALKBH1 expressing cells.

A. Expression of hALKBH1 in mouse Alkbh1^−/− fibroblasts. Lysates of indicated clones (20 µg) were analyzed by Western blot using a monoclonal anti-ALKBH1 antibody. The positions of two molecular mass markers are shown in kDa. B. Growth curves of Alkbh1^−/− fibroblasts and the same cells producing hALKBH1. Cells were plated at low cell density, harvested at the indicated time point by trypsinization and counted. Survival curves for Alkbh1^−/− fibroblasts expressing hALKBH1 or containing a vector control when treated with various concentrations of H₂O₂ (C) or MMS (D). Error bars indicate standard deviations derived from three (H₂O₂) or two (MMS) independent experiments.

Figure 4. Ig class switching to IgG1 is not reduced in Alkbh1^−/− splenic B cells.

A. Flow cytometry results showing surface antibody expression four days after treatment to switch to IgG1. B. Analysis of flow cytometry data related to formation of IgG1 in ALKBH1 deficient splenic B cells normalized to the switching rate of Alkbh1^+/+ B cells. Four mice were used for each genotype and error bars represent standard deviations from these four independent experiments.