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. 2008 Nov;4(11):e1000264.
doi: 10.1371/journal.pgen.1000264. Epub 2008 Nov 21.

Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae

Yong Yang  1 , Joan Sterling, Francesca Storici, Michael A Resnick, Dmitry A Gordenin
Affiliations

Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae

Yong Yang et al. PLoS Genet. 2008 Nov.

Abstract

The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase zeta. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutagenesis in long ssDNA formed during repair of site-specific double-strand break.
(A) Strains to assess DSB-repair-associated mutagenesis. LYS2 and URA3 genes were moved into positions near the forward mutation reporter CAN1 in the left arm of chromosome V (shown not to scale; telomeric repeats are shown as a triangle). A self-generating DSB cassette containing a hygromycin resistance gene, the I-SceI cut site and the ORF for the I-SceI endonuclease under the control of the inducible GAL1-promoter was placed in the middle of the LYS2 (DSB-cen) or the URA3 (DSB-tel) gene. The “no-DSB” control cassette in the LYS2 location contained a non-cleavable I-SceI half-site. (B) Damage-induced mutagenesis in long ssDNA formed at a site-specific DSB. The molecular mechanism of DSB-repair with oligonucleotide is shown as described for the “template” model in . After 5′→3′ resection oligonucleotides anneal to the complementary parts of unresected strands on both sides of DSB. Non-complementary tails of unresected strands (grey lines) are removed and the new 3′-ends annealed to oligonucleotides are used to prime DNA synthesis (short-hatched lines with arrowheads). Second round of 5′→3′ resection removes oligonucleotides allowing repair via single-strand annealing between newly formed complementary ends of a DSB (not shown). Large ss-gaps created by resection should be restored to ds-state in order to accomplish repair. Acute DNA damage (indicated only in ssDNA by “*”) was applied after holding cells for 3 hr or 6 hr following DSB-induction to allow 5′→3′ resection.
Figure 2
Figure 2. UV-mutagenesis associated with DSB-repair.
(A, B) Wild-type and mutant yeast strains were incubated in galactose to induce a DSB for 3 hr (A) or 6 hr (B) and then treated with different doses of UV light. The can1 mutant frequencies were determined among Lys+ (DSB-cen) or Ura+ (DSB-tel) transformants that arose after DSB induction and repair by oligonucleotides. Mutation frequencies determined in cell populations of strains containing the Hyg-Gal-I-SceI insert lacking the I-SceI cut site in the cen (LYS2) position served as “no-DSB” controls. The medians of mutation frequencies obtained in several independent transformation experiments are presented as log10 (mutation frequency×104). The values of median frequencies, ranges of variation and numbers of repeats are provided in Tables S1 and S2. The heights of the bars represent either median frequencies, or the upper limit of 95% CI when the number of mutants was small (indicated with *). Numbers above bars represent frequencies of induced mutations for all variants involving DNA damage or frequencies of spontaneous mutations for “no damage” controls (shown in parentheses).
Figure 3
Figure 3. MMS-mutagenesis associated with DSB-repair in wild type (WT) and various mutants.
Wild-type and mutant yeast strains were incubated in galactose to induce a DSB for 6 hr and then treated with 11.8 mM (0.1%) MMS (C) for 15 or 30 min. See Figure 2 legend for other details.
Figure 4
Figure 4. Mutagenesis in long ssDNA formed at uncapped telomere.
(A) Strain to assess mutagenesis associated with an uncapped telomere. The 34 kb non-essential region between the original telomere and NPR2 at the left end of the chromosome V was removed and the LYS2 forward mutation reporter was inserted in the vicinity of the de novo telomere. (B) Damage-induced mutagenesis in long ssDNA formed at an uncapped telomere. Shifting of cdc13-1 mutants to the non-permissive temperature (37°C) results in the formation of an uncapped telomere followed by G2 arrest and formation of ssDNA. After 6 hr incubation allowing 3′→5′ resection to occur, acute DNA damage (UV-C) was applied to cells (DNA damage is indicated only in ssDNA by “*”) and normal cycling was restored by returning cells to the permissive temperature (23°C).
Figure 5
Figure 5. UV-induced mutagenesis associated with uncapped telomere arrest.
Mutagenesis in the subtelomeric LYS2 associated with uncapped telomere arrest. Frequencies of Lys− mutants were measured in a cdc13-1 strain without additional mutations (WT) or with additional mutations in TLS DNA polymerases. “23°C (G1)” - G1 saturated culture after incubation for 72 h at 23°C permissive temperature. “37°C (G2 arrest)”–G2 cells after additional incubation in fresh YPDA medium for 6 h at 37°C non-permissive temperature. Presented are frequencies of Lys− mutants after UV irradiation (45 J/m2) since the numbers of spontaneous mutants were small. The frequencies and variation for spontaneous as well as UV-induced mutations are shown in Table S3. Frequencies are shown separately for the subtelomeric Lys− mutants (Lys− (lys2 mutants)) and for Lys− mutants containing mutations in genes other than LYS2 (Lys− (LYS2 WT)).
Figure 6
Figure 6. UV-induced and spontaneous mutants associated with DSB-repair and uncapped telomere arrest.
(A) Nucleotide changes in simple base substitutions associated with single and multiple mutant alleles. Bars represent the numbers of simple base substitutions in purine (Pu) or pyrimidine (Py) nucleotides in the unresected strand or in the coding strand, if no resection was expected. Detailed information about DNA sequences and mutation types in all mutant alleles is provided in Tables S4, S5, S6, S7, S8, S9, S10, S11, and S12 and summarized in Tables S16, S17, and S18. All sequenced mutants were from the wild-type (for can1 mutants) or cdc13-1 (for lys2 mutants) strains. All simple base substitutions from can1 mutants induced by 20 and 45 J/m2 in the DSB-cen strains were pooled. Other categories are the same as in Table 1 and in Tables S16, S17, and S18. Statistically significant differences in comparison to the “no-DSB-UV” controls for can1 mutants and to the “no arrest (23°C)” control for subtelomeric lys2 mutants are indicated by “**”. (B, C) Simple base substitutions in multiple mutant alleles. Mutations of CAN1 (B) are summarized from Tables S6, S7, and S8 and S10 and mutations of LYS2 (C) from Table S12. Multiple mutant alleles are shown as lines connecting individual mutations. Substitutions in pyrimidines (purple balls) and substitutions in purines (green squares) are shown above their positions in either CAN1 or LYS2 ORF (bottom lines with the arrowheads). Base substitutions are shown for the unresected strand. Indels and complex mutations are not shown. For the subtelomeric LYS2 reporter only distances from telomere are shown. Since presented can1 mutant alleles were obtained in strains with a DSB-site placed either on telomeric (allele numbers with a prefix “T”) or on centromeric side (numbered without a prefix) of the CAN1, coordinates are given within the ORF. Also shown are the distances of the ORF ends from a break in each type of constructs (DSB-tel or DSB-cen).
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