Research ArticleGENETICS

Mutation at a distance caused by homopolymeric guanine repeats in Saccharomyces cerevisiae

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Science Advances  27 May 2016:
Vol. 2, no. 5, e1501033
DOI: 10.1126/sciadv.1501033
  • Fig. 1 Experimental approach to quantifying mutagenicity of G13+ DNA sequences.

    (A) Poly-G sequences were engineered 4 bp upstream of the URA3 translation start site (the 5′UTR region). The G14-ORF (open reading frame) construct (G14-ORF) allowed detection of loss-of-function mutations in the URA3 reading frame by plating on medium containing 5-fluoroorotic acid (5-FOA), which selects for individual cells that contain mutations that inactivate URA3. The red asterisk indicates the mutation site. (B) Using a weak URA3 allele (URA3-w), this construct (G14-repeat) facilitated the detection of the polyguanine repeat expansion mutation, because the mutation, G14 to G15 or longer, results in the 5′-FOA–resistant phenotype. wt, wild type.

  • Fig. 2 Polyguanine sequences cause a localized, directional effect on mutation rate.

    (A) Mutation rates of homopolymeric guanine repeat sequences of increasing length. The estimated phenotypic mutation rate of G0-ORF, G13-ORF, and G14-ORF is 5.4 × 10−7, 13.5 × 10−7, and 20.3 × 10−7, respectively. G11 and G12 had no detectable increase in mutation rate. (B) Mutation rate was measured using CAN1, at a site distal from the URA3 locus. (C) The G14 repeat does not cause an increase in mutation rate if engineered on the template strand (C14-URA3) or on either the coding strand (ORF-G14) or the template strand (ORF-C14) downstream of the URA3 terminator sequence. Significant differences were calculated using t tests. *P < 0.05; **P < 0.005. Error bars show 95% confidence intervals.

  • Fig. 3 Mutational spectrum of 183 5′-FOA–resistant mutants at the URA3 locus.

    5′-FOA–resistant mutants were collected from G0-URA3 (brown) and G14-ORF (blue) strains. Each point mutation is shown directly above the wild-type sequence, and indels and complex mutations are shown directly below.

  • Fig. 4 The effect of G14 mutagenicity is correlated with DNA replication timing.

    (A and B) The regression of mutation rate and replication timing for G14-ORF alleles inserted at six different sites on chromosome XII and two sites on chromosome XV (Spearman’s R = 0.89, P = 0.007) (A) and for insertion of G0-URA3 at the same sites (Spearman’s R = 0.63, P = 0.086) (B). Replication timing is shown in minutes after the release of cells into synchronized S phase, as reported by Nieduszynski et al. (30). Error bars show 95% confidence intervals.

  • Fig. 5 G13 repeats are implicated in the accumulation of replication fork intermediates.

    (A) 2D gels for tet-G0-URA3 and tet-G13-URA3 under the control of a repressible tetR promoter (65) for time points taken at 15, 30, 45, and 60 min after synchronized cells were released from G1 cell cycle arrest. (B) The circled area show replication fork intermediates that are putatively stalled at the beginning of URA3 (Y molecules, blue circle). The reference region (red hexagon) is used to normalize between samples and determine the relative amounts of Y molecules in each treatment. (C) When transcription is enabled (−dox), the G13 construct has ~2.5 as much putative fork stalling as the G0 construct (calculated at the 30-min time point). When transcription is repressed, both G13 and G0 constructs appeared to have more Y molecules, although G13 has only ~1.13 as much replication fork intermediates as G0 (calculated at the 30-min time point). (D) Mutation rates for G0-URA3 and G13-ORF under the control of a repressible tetR promoter. −dox indicates the high-expression treatment, whereas +dox indicates repression of transcription. Significant differences were calculated using t tests; *P < 0.05. Error bars show 95% confidence intervals.

  • Fig. 6 Expansion of polyguanine repeats occurs at a high rate and is RAD52-dependent.

    (A) Mutation rates of G0 and G14-ORF strains compared to their respective rev1 deletion mutants. (B) Mutation rates of G0, G14-repeat, and G14-ORF strains, shown relative to the G0 mutation rate. (C and D) In each panel, the mutation rates of deletion mutants are shown relative to their respective nondeletion progenitor, either G0 (C), G14-ORF (C), or G14-repeat (D). Significant differences calculated using t tests are indicated by asterisks: *P < 0.05 and **P < 0.005. Error bars represent 95% confidence intervals.

  • Fig. 7 Model for the outcome of G13+-induced replication fork stalling.

    (I) The replication fork can proceed in either direction, but the transcription complex must encounter the G13+ repeat before the transcribed gene. (II) Transcription stalls at the G13+ sequence. (III) The replication fork stalls at the G13+ sequence; stalling is more likely if there is a stalled transcription complex already present. (IV) The replication fork detaches from the template and reinitiates replication downstream, leaving a patch of singlestranded DNA that is 800 to 3000 bp in length. (V) The DNA complementary to the single-stranded gap is synthesized using either Rad52-dependent HR (detected using the G14-repeat construct) or Rev1-dependent TLS (detected using the G14-ORF construct) to bypass the difficult-to-replicate region.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/5/e1501033/DC1

    fig. S1. Expansion of the polyguanine repeat (G14 to G15) reduces the Ura3 protein abundance but not the mRNA level.

    fig. S2. G11 to G14 sequences do not stop DNA polymerase from synthesizing DNA, whereas G-quadruplex does.

    table S1. Summary table of 318 sequenced ura3 mutants from G0, G14-ORF, and G14-repeat strains.

    table S2. Chromosome insertion position and replication timing for engineered G14-URA inserts.

    References (66, 67)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Expansion of the polyguanine repeat (G14 to G15) reduces the Ura3 protein abundance but not the mRNA level.
    • fig. S2. G11 to G14 sequences do not stop DNA polymerase from synthesizing DNA, whereas G-quadruplex does.
    • Legend for table S1
    • table S2. Chromosome insertion position and replication timing for engineered G14-URA inserts.
    • References (66, 67)

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Summary table of 318 sequenced ura3 mutants from G0, G14-ORF, and G14-repeat strains.

    Files in this Data Supplement:

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