Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells - PubMed (original) (raw)

Non-homologous end joining as an important mutagenic process in cell cycle-arrested cells

Erich Heidenreich et al. EMBO J. 2003.

Abstract

Resting cells experience mutations without apparent external mutagenic influences. Such DNA replication-independent mutations are suspected to be a consequence of processing of spontaneous DNA lesions. Using experimental systems based on reversions of frameshift alleles in Saccharomyces cerevisiae, we evaluated the impact of defects in DNA double-strand break (DSB) repair on the frequency of replication-independent mutations. The deletion of the genes coding for Ku70 or DNA ligase IV, which are both obligatory constituents of the non-homologous end joining (NHEJ) pathway, each resulted in a 50% reduction of replication-independent mutation frequency in haploid cells. Sequencing indicated that typical NHEJ-dependent reversion events are small deletions within mononucleotide repeats, with a remarkable resemblance to DNA polymerase slippage errors. Experiments with diploid and RAD52- or RAD54-deficient strains confirmed that among DSB repair pathways only NHEJ accounts for a considerable fraction of replication-independent frameshift mutations in haploid and diploid NHEJ non-repressed cells. Thus our results provide evidence that G(0) cells with unrepressed NHEJ capacity pay for a large-scale chromosomal stability with an increased frequency of small-scale mutations, a finding of potential relevance for carcinogenesis.

PubMed Disclaimer

Figures

None

Fig. 1. Frequencies of replication-independent Lys+ revertant colonies in a haploid repair-proficient (wild type) and two DSB repair-deficient strains with deletions of RAD52 or RAD54, respectively. A time course starting with day 4 after plating is shown because earlier arising colonies were considered as originating (surely or potentially) from pre-existing revertants. Cumulative colony counts were normalized to the numbers of cells on the plates. The mean of three to four experiments is shown together with standard error bars.

None

Fig. 2. Frequencies of replication-independent revertant colonies in haploid NHEJ-deficient strains. Two different frameshift alleles were analysed. (A) Reversion of _lys2_Δ_BglII_-caused lysine auxotrophy. (B) Reversion of _hom3-10_-caused methionine auxotrophy. Graph style as described for Figure 1.

None

Fig. 3. Sequence analysis of lys2_Δ_BglII reversions in the haploid DNA ligase IV-deficient strain. Only the relevant part of the LYS2 allele from nucleotides 362 to 507 is shown (numbered relatively to the translation start). Reverting frameshifts of the _lys2_Δ_BglII_-specific GATC duplication (bold) must take place in the represented sequence stretch between two out-of-frame stop codons (underlined) with respect to the wild-type reading frame. Fifty-six intragenic proliferation-dependent reversions are indicated above the sequence, and 59 intragenic replication-independently generated reversions are displayed below the sequence. Symbols: –1 deletions are shown by Δ; inserted nucleotides are shown together with arrow heads; individual complex sequence alterations (e.g. deletions accompanied by base substitutions) are displayed in parentheses.

None

Fig. 4. Statistical analysis of reversion sequence spectra. The similarities of pairs of sequence datasets of haploid wild-type or DNA ligase IV-deficient revertants were calculated using the algorithm of Adams and Skopek, available as a computer program (Cariello et al., 1994). Significance levels are indicated as follows: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., no significant difference. Wild-type sequence data are derived from Heidenreich and Wintersberger (2001).

None

Fig. 5. Frequencies of replication-independent Lys+ revertant colonies in diploid strains. (A) NHEJ-deficient strains. (B) Strains deficient in homology-dependent DSB repair pathways, together with wild-type strains differing in mating type properties. Wild-type a/a is repair proficient and homozygous for the mating type allele MATa. All other strains are MATa/α. Graphs are drawn to the same scale; style as described for Figure 1.

Similar articles

Cited by

References

    1. Åström S.U., Okamura,S.M. and Rine,J. (1999) Yeast cell-type regulation of DNA repair. Nature, 397, 310. - PubMed
    1. Babudri N., Pavlov,Y.I., Matmati,N., Ludovisi,C. and Achilli,A. (2001) Stationary-phase mutations in proofreading exonuclease-deficient strains of the yeast Saccharomyces cerevisiae. Mol. Genet. Genomics, 265, 362–366. - PubMed
    1. Baranowska H., Policinska,Z. and Jachymczyk,W.J. (1995) Effects of the CDC2 gene on adaptive mutation in the yeast Saccharomyces cerevisiae. Curr. Genet., 28, 521–525. - PubMed
    1. Cairns J., Overbaugh,J. and Miller,S. (1988) The origin of mutants. Nature, 335, 142–145. - PubMed
    1. Cariello N.F., Piegorsch,W.W., Adams,W.T. and Skopek,T.R. (1994) Computer program for the analysis of mutational spectra: application to p53 mutations. Carcinogenesis, 15, 2281–2285. - PubMed

Publication types

MeSH terms

LinkOut - more resources