Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 1;72(3):583-593.e4.
doi: 10.1016/j.molcel.2018.08.036. Epub 2018 Oct 4.

Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles

Andrés Mansisidor  1 , Temistocles Molinar Jr  1 , Priyanka Srivastava  1 , Demetri D Dartis  1 , Adriana Pino Delgado  1 , Hannah G Blitzblau  2 , Hannah Klein  3 , Andreas Hochwagen  4
Affiliations

Genomic Copy-Number Loss Is Rescued by Self-Limiting Production of DNA Circles

Andrés Mansisidor et al. Mol Cell. .

Abstract

Copy-number changes generate phenotypic variability in health and disease. Whether organisms protect against copy-number changes is largely unknown. Here, we show that Saccharomyces cerevisiae monitors the copy number of its ribosomal DNA (rDNA) and rapidly responds to copy-number loss with the clonal amplification of extrachromosomal rDNA circles (ERCs) from chromosomal repeats. ERC formation is replicative, separable from repeat loss, and reaches a dynamic steady state that responds to the addition of exogenous rDNA copies. ERC levels are also modulated by RNAPI activity and diet, suggesting that rDNA copy number is calibrated against the cellular demand for rRNA. Last, we show that ERCs reinsert into the genome in a dosage-dependent manner, indicating that they provide a reservoir for ultimately increasing rDNA array length. Our results reveal a DNA-based mechanism for rapidly restoring copy number in response to catastrophic gene loss that shares fundamental features with unscheduled copy-number amplifications in cancer cells.

Keywords: Fob1; Hmo1; copy-number variations; eccDNA; genome instability; rDNA; rRNA genes.

PubMed Disclaimer

Conflict of interest statement

Declaration of Interests:

The authors declare no competing interests.

Figures

Figure 1:
Figure 1:. ERC levels anti-correlate with chromosomal rDNA array size.
(A) Random deletion of chromosomal rDNA repeats using a URA3-targeting plasmid containing homologies to the rDNA intergenic sequence (orange and pink). (B) PFGE analysis of deletion strains. Southern probe (URA3) detects the ura3 locus (Chr.5) and the URA3 insertion in rDNA (Chr. 12). (C) Southern analysis of ERCs probing against rDNA sequence and GAT1 (loading control) after digestion by AfeI. ERC signals labeled “a-f” based on electrophoretic mobility. (D) Total ERC signal (bands “a-e”) relative to GAT1 as function of array size; P < 4.5×10‒5 (Spearman’s rank correlation) and r2 fit to a power law function. Band f was not quantified due to proximity to wells, which is subjected to non-reproducible smearing and cracking during transfer. (E) Southern analysis of total ERCs with and without digestion by exonuclease RecBCD.
Figure 2:
Figure 2:. URA3-marked ERCs anti-correlate with array size, but not with marker loss.
(A-B) Southern analysis of URA3-marked ERCs as function of array size after digestion with NdeI. (C) 3.5-fold dilutions of rDNA deletion strains spotted onto synthetic complete (SC) media with and without 5-FOA. (D-E) Southern analysis of multi-copy, URA3-marked ERCs and quantification relative to single-copy ERCs. Data are represented as mean of n=3 clonal replicates ± standard deviation. Exonuclease RecBCD was used to digest linear DNA fragments, then heat inactivated before subsequent digestion with NdeI.
Figure 3:
Figure 3:. ERC accumulation responds to copy number.
(A-D) Southern analysis of ERCs in ~90-repeat strains. (A-B) Kinetics of ERC accumulation (bands a-e) relative to GAT1 after FOB1 induction with 100nM beta-estradiol. (C-D) ERC levels (bands a, c, d, e) relative to GAT1 after introduction of high-copy (2) rDNA plasmids or after further increasing plasmid copy number (leu2); ERC b (#) overlapped with plasmid signal and was not quantified. Data are represented as mean of n=4 biological replicates ± standard deviation. **P < 0.01, ***P < 0.001 (one-tailed t-test with Holm correction).
Figure 4:
Figure 4:. Glycerol metabolism decreases ERC levels to a new steady state.
(A-D) Southern analysis of ERCs in ~90-repeat strains after shift from dextrose (YPD, t=0) to glycerol media. (A) Representative Southern blot of total ERCs. (B) Average signal of biological replicates, n=4 (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Representative Southern blot of URA3-marked ERCs. (D) Average signal of biological replicates, n=4, performed in duplicate (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 5:
Figure 5:. ERC formation is linked to rRNA levels.
(A) Quantification of ERC levels in ~120-repeat strains (bands a-e; URA3 probe) relative to GAT1 upon deletion of HMO1 (n=4), RPA49 (n=6), or UAF30 (n=4); data are represented as mean ± standard deviation *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t-test with Holm correction; all experiments represent biological replicates). (B-C) URA3-marked ERCs in WT and hmo1Δ mutants by arrays size after digestion by NdeI; data are represented as mean ± standard deviation, **P < 0.01 (two-tailed t-test; clonal replicates n=3). WT data in (C) is same as in Figure 2B.
Figure 6:
Figure 6:. Copy number alters Fob1 binding and ERC reinsertion.
(A-C) ChIP-qPCR analysis of Fob1 levels in the rDNA (A) and at the RFB (B, C) in indicated strains and conditions; data are represented as mean ± standard deviation, *P < 0.05, **P < 0.01 (two-tailed t-test; biological replicates, n=3). (D) Strategy for detecting cells with multiple URA3 insertions (green) by targeting URA3 with a LEU2 construct (blue). (E) leu+ ura+ transformants relative to the total leu+ transformants in the indicated strains; data are represented as mean ± standard deviation, *P < 0.05, **P < 0.01 (pair-wise, two-tailed t-tests with Holm correction; clonal replicates, n=3).
Figure 7:
Figure 7:. Model for Fob1-dependent ERC formation.
(A-E) Model for ERC formation that was adapted from (Vogt et al., 2004) and modified to include the unipolar directionality of rDNA replication (Brewer and Fangman, 1988). (A) Asterisks represent origin firing, which typically occurs in a clustered fashion (Pasero et al., 2002). (B) Rightward moving forks are stalled at the Fob1 binding site (RFB), which is vulnerable to collapse and DSB formation. (C) When neighboring RFBs collapse into DSBs, newly synthesized DNA may be excised and (D) circularize through the stand-annealing activity of Rad52. Passive replication from the rightmost fork could then re-replicate repeats that have formed ERCs, (E) ultimately amplifying rDNA copy number.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albert B, Colleran C, Léger-Silvestre I, Berger AB, Dez C, Normand C, Perez-Fernandez J, McStay B, Gadal O (2013). Structure-function analysis of Hmo1 unveils an ancestral organization of HMG-Box factors involved in ribosomal DNA transcription from yeast to human. Nucleic Acids Res. 41, 10135–10149. doi:10.1093/nar/gkt770 - DOI - PMC - PubMed
    1. Aldrich JC, Maggert KA (2015). Transgenerational Inheritance of Diet-Induced Genome Rearrangements in Drosophila. PLOS Genet. 11, e1005148. doi:10.1371/journal.pgen.1005148 - DOI - PMC - PubMed
    1. Averbeck KT, Eickbush TH (2005). Monitoring the mode and tempo of concerted evolution in the Drosophila melanogaster rDNA locus. Genetics 171, 1837–1846. doi:10.1534/genetics.105.047670 - DOI - PMC - PubMed
    1. Benjamin KR, Zhang C, Shokat KM, Herskowitz I (2003). Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2. Genes Dev. 17, 1524–1539. doi:10.1101/gad.1101503 - DOI - PMC - PubMed
    1. Blake D, Luke B, Kanellis P, Jorgensen P, Goh T, Penfold S, Breitkreutz BJ, Durocher D, Peter M, Tyers M (2006). The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae. Genetics 174, 1709–1727. doi:10.1534/genetics.106.057836 - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources