Research ArticleGENETICS

Impaired cohesion and homologous recombination during replicative aging in budding yeast

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Science Advances  07 Feb 2018:
Vol. 4, no. 2, eaaq0236
DOI: 10.1126/sciadv.aaq0236
  • Fig. 1 Transcription of ncRNA from the rDNA during aging.

    (A) Structure of yeast rDNA locus. (B) Strand-specific reverse transcription quantitative PCR analysis from both the Watson (+) and Crick (−) strands of NTS1/2 during aging. RNA levels were normalized to ACT1 before normalizing to 1 for young cells in each case. The average and SEM of three replicates are plotted. *P < 0.05, as determined by Student’s t test.

  • Fig. 2 Cohesion is lost during replicative aging.

    (A) ChIP analysis of hemagglutinin (HA)–tagged Mcd1 in young and old cells. An untagged strain was used as a negative control. The average and SEM of four replicates are plotted. *P < 0.05, as determined by Student’s t test. (B) Loss of cohesion at rDNA and centromeric regions with aging. Representative images are shown on the left. The average and SEM of three replicates are plotted. The arrows indicate one spot or two spots of GFP lac repressor. *P < 0.05, as determined by Student’s t test.

  • Fig. 3 NTS1/2 transcription does not directly correlate with cohesin association.

    (A) ChIP analysis of Mcd1 occupancy in log-phase cultures. Data shown were normalized to a positive control region and then to E-pro. The average and SEM of two replicates are plotted. (B) Strand-specific qRT-PCR analysis, as in Fig. 1B, in WT and fob1Δ strains. (C) Western analysis of HA-tagged Mcd1 and (D) tandem affinity purification (TAP)–tagged Smc1, Smc3, and Scc3 with untagged strain as a control. Histone H3 protein level is known to be decreased in old cells (52), whereas Rad52 protein level remains constant during aging (this study). Antibodies against TAP or protein A were used, as indicated.

  • Fig. 4 The rDNA locus and chr XII become increasingly unstable with age.

    (A) Analysis of yeast chromosomes by PFGE. Left panel: SYBR-safe staining. Right panel: Southern hybridization using a chr XII (rDNA) probe. The rad52Δ mutant was used as a control with a different rDNA copy number. (B) As in (A), probes to the rDNA or the left arm of chr XII on the same samples. (C) Left panel: SYBR-safe staining of same number of cells analyzed by PFGE. Right panel: Southern blot using the indicated probe. Closed triangles indicate stuck DNA in the well. Quantification of the relative intensity of stuck DNA in old cells compared to young cells is shown below with the average and SEM of three replicates. *P < 0.05, significant change, as determined by Student’s t test. (D) The DSB at the RFB is not more abundant in old cells. Schematic showing use of Bgl II digestion to detect DSB at RFB. Southern detection using either the DSB probe or the control probe is shown. The asterisk indicates DNA fragments generated from DSBs near the RFB, whereas the arrows indicate fragments from restriction digestion with no DSB at RFB.

  • Fig. 5 The aging genome accumulates random damage, whereas rDNA instability appears to promote global genomic instability.

    (A) As in Fig. 4A using the same numbers of young and old WT yeast cells probed for three chromosomes of different sizes: chr IV (long), chr X (middle-sized), and chr I (short). Left panels: SYBR-safe staining. Right panels: Southern blots. The graph shows percent loss of band intensity in aged cells measured from Southern blots, following normalizing signal intensity of young cells to 100% for each chromosome. The average and SEM of three replicates are plotted. The asterisk indicates significant change in chromosomal band intensities between chromosomes of different lengths in old cells (P < 0.05), as determined by Student’s t test. (B) Old cells have more global DNA damage than young cells, and this is partially restored in FOB1 deleted cells, as determined by flow cytometry analysis. Top panel: Levels of fluorescein-labeled dUTP in the absence of TdT indicating the background incorporation. Bottom panel: Levels of fluorescein-labeled dUTP in the presence of TdT that indicates levels of DNA damage. For both panels, dotted lines indicate young cells, and solid lines indicate old cells. (C) PFGE analysis from same numbers of young and old yeast cells from both WT and fob1Δ strains. The net percent loss of chromosomal band intensities during aging was measured from quantifying band intensities of all chromosomes from SYBR-safe stained agarose gels. The averages of two replicates are plotted here, along with the best-fit line for each group. The R2 value for WT is 0.39911, and for fob1Δ, the value is 0.72577. Individual experimental data are shown in fig. S6. (D) Similar analysis as in (A), with cells from both WT and fob1Δ strains. In each case, the left panel shows SYBR gold staining of chromosomes, and right panel shows Southern hybridization using the indicated probe.

  • Fig. 6 Aged cells have impaired DSB repair due to reduced repair protein levels.

    (A) PFGE analysis of chromosomes from the same numbers of young and old WT yeast cells. Samples were collected before damage induction (Unt), after MMS treatment (MMS), and after MMS removal and recovery at 3 and 6 hours, respectively. (B and C) Western blot analysis to measure key DNA repair protein levels during aging. Samples are loaded according to equal cell numbers. (B) Proteins using commercial antibodies. (C) HA-tagged proteins, with untagged strain as additional control. Ponceau staining is shown to show equal loading. (D) Quantification of percentage of young and old cells with detectable γH2A foci in both WT and single-copy Rad51-overexpression (OE) strains. The average and SEM of three replicates are plotted. *P < 0.05, as determined by Student’s t test. (E) As in (D), but for Mre11-OE. (F) As for (A) in old WT and old Rad51-overexpression yeast cells. (G) As in (F), but for Mre11-OE.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/2/eaaq0236/DC1

    fig. S1. No change in rRNA levels during aging.

    fig. S2. Cohesin occupancy is not affected by NTS1 transcription, but cohesins are reduced during aging.

    fig. S3. Appearance of two major chromosomal bands containing rDNA during aging.

    fig. S4. The two major chromosomal bands containing rDNA appear to comprise mainly rDNA repeats in old cells.

    fig. S5. Increased chromosomal instability proportional to chromosome length and rDNA instability is observed during aging in WT cells.

    fig. S6. The DNA damage checkpoint is intact in old cells.

    fig. S7. Rad51 and Mre11 overexpression.

    fig. S8. Model for rDNA instability and reduced HR causing global genomic instability to limit replicative life span.

    table S1. Yeast strains used in this study.

    table S2. Primers used in this study.

    Reference (53)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. No change in rRNA levels during aging.
    • fig. S2. Cohesin occupancy is not affected by NTS1 transcription, but cohesins are reduced during aging.
    • fig. S3. Appearance of two major chromosomal bands containing rDNA during aging.
    • fig. S4. The two major chromosomal bands containing rDNA appear to comprise mainly rDNA repeats in old cells.
    • fig. S5. Increased chromosomal instability proportional to chromosome length and rDNA instability is observed during aging in WT cells.
    • fig. S6. The DNA damage checkpoint is intact in old cells.
    • fig. S7. Rad51 and Mre11 overexpression.
    • fig. S8. Model for rDNA instability and reduced HR causing global genomic instability to limit replicative life span.
    • table S1. Yeast strains used in this study.
    • table S2. Primers used in this study.
    • References (5, 19, 53)

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