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. 2005 Jun 15;24(12):2205-13.
doi: 10.1038/sj.emboj.7600689. Epub 2005 May 19.

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

Qian An  1 , Peter Robins, Tomas Lindahl, Deborah E Barnes
Affiliations

C --> T mutagenesis and gamma-radiation sensitivity due to deficiency in the Smug1 and Ung DNA glycosylases

Qian An et al. EMBO J. .

Abstract

The most common genetic change in aerobic organisms is a C:G to T:A mutation. C --> T transitions can arise through spontaneous hydrolytic deamination of cytosine to give a miscoding uracil residue. This is also a frequent DNA lesion induced by oxidative damage, through exposure to agents such as ionizing radiation, or from endogenous sources that are implicated in the aetiology of degenerative diseases, ageing and cancer. The Ung and Smug1 enzymes excise uracil from DNA to effect repair in mammalian cells, and gene-targeted Ung(-/-) mice exhibit a moderate increase in genome-wide spontaneous mutagenesis. Here, we report that stable siRNA-mediated silencing of Smug1 in mouse embryo fibroblasts also generates a mutator phenotype. However, an additive 10-fold increase in spontaneous C:G to T:A transitions in cells deficient in both Smug1 and Ung demonstrates that these enzymes have distinct and nonredundant roles in suppressing C --> T mutability at non-CpG sites. Such cells are also hypersensitive to ionizing radiation, and reveal a role of Smug1 in the repair of lesions generated by oxidation of cytosine.

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Figures

Figure 1
Figure 1
siRNA-mediated downregulation of Smug1 in MEF cell lines. (A) Smug1 mRNA level was detected by real-time PCR of reverse-transcribed cDNA from MEFs: wild-type (open bar), Ung−/− (diagonal striped bar), Smug1↓ (solid bar) and Smug1↓, Ung−/− (cross-hatched bar). Error bars show the standard error of the mean from three experiments. (B) Uracil-DNA glycosylase activity was assayed on a 19-mer double-stranded oligonucleotide with a centrally placed uracil residue in the 5′-32P-end-labelled strand (lane 1) in MEF nuclear extracts supplemented with AP endonuclease. The 9-mer radiolabelled product was resolved by denaturing PAGE and detected by phosphorimager. The substrate was incubated with wild-type extract (WT; lanes 2–5) or Smug1-deficient extracts from Smug1↓ and Smug1↓, Ung−/− cell lines (lanes 6–10), after preincubation with the UNG inhibitor Ugi and/or SMUG1 antibodies, as indicated.
Figure 2
Figure 2
Survival of Smug1-deficient MEF cell lines after exposure to γ-irradiation. Wild-type (filled square), Smug1↓ (open square), Ung−/− (filled triangle) and Smug1↓, Ung−/− (open triangle) MEF cell lines were treated at the doses indicated and surviving colonies scored after 10–12 days as a percentage of untreated control. All values are the mean from three experiments; error bars show the standard error of the mean.
Figure 3
Figure 3
Structures of cytosine, uracil, thymine nucleobases and relevant oxidized derivatives. Lesions excised by UNG and/or SMUG1 are indicated.
Figure 4
Figure 4
Cytosine-derived base lesions excised from irradiated DNA by human DNA glycosylases. HPLC profiles are shown of ethanol-soluble radiolabelled material released from γ-irradiated [3H]cytosine-containing DNA by the uracil-DNA glycosylases UNG (filled triangle) and SMUG1 (open square); SMUG1 was preincubated with the UNG inhibitor Ugi. The positions of reference compounds are indicated (detected by UV absorbance).
Figure 5
Figure 5
Mutational spectra in MEF cell lines deficient in Smug1 and/or Ung. The hprt open reading frame (672 bp) was sequenced in individual mutant colonies (as in Table I) from wild-type (WT; open bar), Ung−/− (diagonal striped bar), Smug1↓ (solid bar) and Smug1↓, Ung−/− (cross-hatched bar) MEF cell lines. Data are presented for the different classes of mutation (as indicated on the x-axis). The number of individual mutations of each class (N) and the total number of mutations are shown below for each cell line; the number of mutant clones sequenced (M) is given in parentheses. The frequency of each class of mutation (y-axis) is calculated as N/(672M).
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