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. 2012 May 1;11(5):502-10.
doi: 10.1016/j.dnarep.2012.02.005. Epub 2012 Mar 17.

Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts

Stephanie L Nay  1 , Dong-Hyun Lee, Steven E Bates, Timothy R O'Connor
Affiliations

Alkbh2 protects against lethality and mutation in primary mouse embryonic fibroblasts

Stephanie L Nay et al. DNA Repair (Amst). .

Abstract

Alkylating agents modify DNA and RNA forming adducts that disrupt replication and transcription, trigger cell cycle checkpoints and/or initiate apoptosis. If left unrepaired, some of the damage can be cytotoxic and/or mutagenic. In Escherichia coli, the alkylation repair protein B (AlkB) provides one form of resistance to alkylating agents by eliminating mainly 1-methyladenine and 3-methylcytosine, thereby increasing survival and preventing mutation. To examine the biological role of the mammalian AlkB homologs Alkbh2 and Alkbh3, which both have similar enzymatic activities to that of AlkB, we evaluated the survival and mutagenesis of primary Big Blue mouse embryonic fibroblasts (MEFs) that had targeted deletions in the Alkbh2 or Alkbh3 genes. Both Alkbh2- and Alkbh3-deficient MEFs were ∼2-fold more sensitive to methyl methanesulfonate (MMS) induced cytotoxicity compared to the wild type control cells. Spontaneous mutant frequencies were similar for the wild type, Alkbh2-/- and Alkbh3-/- MEFs (average--1.3×10(-5)). However, despite the similar survival of the two mutant MEFs after MMS treatment, only the Alkbh2-deficient MEFs showed a statistically significant increase in mutant frequency compared to wild type MEFs after MMS treatment. Therefore, although both Alkbh2 and Alkbh3 can protect against MMS-induced cell death, only Alkbh2 shows statistically significant protection of MEF DNA against mutations following treatment with this exogenous methylating agent.

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Conflict of interest statement

Conflict of interest Statement

The authors declare there are no conflicts of interest.

Figures

Figure 1
Figure 1
Maps for targeted deletions of mouse α-ketoglutarate/Fe(II) dioxygenases Alkbh2 and Alkbh3 were generated as follows: (a) Alkbh2 and the site targeting the Mus musculus genomic sequence located on mouse chromosome 5 (LOCUS NC_000071 from the NCBI and Mouse Genome Sequencing Consortium). (b) Southern blots for Alkbh2+/− heterozygotes using a probe for the 5’arm. +/+ is a WT control and +/− are different clones identified as heterozygotes by initial PCR screening. (c) Southern blots for Alkbh2+/− heterozygotes using a probe for the 3’arm. +/+ is a WT control and +/− are different clones identified as heterozygotes by initial PCR screening. Clones used to probe (b) and (c) are identical. (d) Alkbh3 and the site targeting the Mus musculus genomic sequence located on mouse chromosome 2 (LOCUS NC_000068 from the NCBI and Mouse Genome Sequencing Consortium). (e) Southern blots for Alkbh3+/− heterozygotes using a probe for the 5’arm. +/+ is a WT control and +/− are for different clones identified as heterozygotes by initial PCR screening. (f) Southern blots for Alkbh3+/− heterozygotes using a probe for the 3’arm. +/+ is a WT control and +/− are for different clones identified as heterozygotes by initial PCR screening. Clones used to probe (e) and (f) are identical.
Figure 2
Figure 2
PCR confirmation of Alkbh2 and Alkbh3 targeted deletions was determined by amplification of deleted or inserted sequences in the Alkbh2 or Alkbh3 gene, using PCR. (a) The Exon 4 region of Alkbh2, the exon 7 region of Alkbh3 and Neo gene contained in the cassette replacing AlkBh2 or AlkBh3 exons were amplified from genomic DNA extracted from primary WT and Alkbh2 or Alkbh3 knock-out MEFs. The 199 bp band is the product from Exon 4 of a functional Alkbh2. The 160 bp band is the product from Exon 7 of a functional Alkbh3. The 171 bp band is the neomycin PCR product found in the cassette used for targeted deletion of Alkbh2 or Alkbh3.
Figure 3
Figure 3
Cell survival of WT, Alkbh2−/−, or Alkbh3−/− primary Big Blue MEFs was determined 48 h post-treatment with MMS, as described in Experimental Procedures. (a) Survival was monitored using Trypan Blue staining to determine the percentage of intact cellular membranes in each culture. (b) IC50 values for all the primary MEFs. All samples were analyzed in triplicate and with curve fitting and statistical analysis performed using Prism software. *** P equals 0.0003 and **P equals 0.003, compared to WT IC50.
Figure 4
Figure 4
MMS-induced mutant frequencies for WT and Alkbh2−/− or Alkbh3−/− primary MEFs. Frequencies were determined by subtracting the spontaneous mutant frequency, as determined in G1250 cells infected with λLIZ phage containing packaged cII genomic DNA, isolated from primary WT, Alkbh2, and Alkbh3 knock-out MEFs, from the observed mutant frequency following 4 mM MMS treatment. *** P equals 0.0001.
Figure 5
Figure 5
Spontaneous and 4 mM MMS-induced mutation spectrum map from WT primary MEFs. 29 spontaneous and 36 MMS-induced mutants were sequenced. A ‘v’ indicates an insertion mutation. The exact number of each mutation type can be found in Table 2.
Figure 6
Figure 6
Spontaneous and 4 mM MMS-induced mutation spectrum map from Alkbh2−/− primary MEFs. 54 spontaneous and 44 MMS-induced mutants were sequenced. A ‘v’ indicates an insertion mutation. The exact number of each mutation type can be found in Table 2.
Figure 7
Figure 7
Spontaneous and 4 mM MMS-induced mutation spectrum map from Alkbh3−/− primary MEFs. 43 spontaneous and 57 MMS-induced mutants were sequenced. A ‘v’ indicates an insertion mutation. The exact number of each mutation type can be found in Table 2.
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