Skip to main page content
U.S. flag
See More

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
. 2011 Dec 23;44(6):978-88.
doi: 10.1016/j.molcel.2011.10.017.

RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability

Lamia Wahba  1 , Jeremy D Amon, Douglas Koshland, Milena Vuica-Ross
Affiliations

RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability

Lamia Wahba et al. Mol Cell. .

Abstract

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA:DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3Δ, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA:DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNase H act as guardians of the genome.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1. Monitoring GCRs on the YAC
Assay for gross chromosomal rearrangements. Rates are determined by plating 105 cells and determining the number of cells that retained the YAC (His+) but lost the telomeric marker (Ura-), or lost the YAC completely (His- and Ura-).
FIGURE 2
FIGURE 2. Rates of YAC instability in various mutant classes without and with RNAse H over-expression
(a) RNA biogenesis mutants carrying the YAC were assayed for GCRs with either an empty vector (black bars) or a 2μ plasmid expressing RNase H1 (grey bars). Error bars represent standard deviation calculated from at least 4 independent colonies. (b) Repair mutants carrying the YAC were assayed for GCRs with either an empty vector (black bars) or a 2μ plasmid expressing RNase H1 (grey bars). Error bars represent standard deviation calculated from at least 4 independent colonies.
FIGURE 3
FIGURE 3. Rates of chromosome III and XII instability in transcription mutants
(a) RNA biogenesis mutants with a modified chromosome III were assayed for GCRs. Error bars represent standard deviation calculated from at least 4 independent colonies (b) Percent of chromosome XII instability in sin3Δ mutants. Percent instability is determined by plating on YPD plates and determining the number of colonies with red sectors. A total of 13,000 to 26,000 cells originating from 20 independent colonies were scored for each genotype. Confidence intervals (CI) are calculated from the standard deviation of the 20 independent colonies.
FIGURE 4
FIGURE 4. Cytological detection of RNA-DNA hybrids in transcription and RNase H mutants
(a) Representative images of chromatin spreads stained with S9.6 antibody, recognizing RNA-DNA hybrids. (b) Measurement distribution of staining intensity in WT, sin3Δ, and rnh1Δrnh201Δ. A total of 100 cells were analyzed for each genotypic class.
FIGURE 5
FIGURE 5. RNA-DNA hybrids and genome instability at the rDNA locus in sin3Δ
(a) Left panel- Representative images for patterns of staining observed with the RNA-DNA hybrid antibody. Arrow heads point to the nucleolus. Right panel- Distribution of rDNA locus enrichment in WT, sin3Δ, rnh1Δrnh201Δ and med13Δ. Only nuclei with an intensity of 20,000 or more were scored. n= 25, 100, 100, and 50 for each genotype, respectively. (b) Transcription mutants with URA3 integrated at the rDNA were assayed for loss of the URA3 marker. Error bars represent standard deviation calculated from at least 8 independent colonies. (c) Representative images of chromatin spreads stained with S9.6 antibody, recognizing RNA-DNA hybrids. Strains were harboring either an empty control 2μ plasmid or one expressing RNase H1.
FIGURE 6
FIGURE 6. The absence of Sin3p increases the number of cells with Rad52-GFP foci
(a) The number of cells with Rad52-GFP foci was quantified in asynchronous log phase cells of WT and sin3Δ mutant carrying either an empty control 2μ plasmid or one expressing RNase H1. A total of approximately 1000 cells were scored for each of the two WT genotypes and 3000 for each sin3Δ genotype, spanning three or more experiments of two independently isolated colonies. For all graphs, error bars represent standard deviation between experiments. (b) The number of cells with Rad52-GFP foci was quantified in sin3Δ cells carrying the empty of RNase H1 plasmid at indicated time points post-release from G1 arrest. A total of 600-1000 cells were scored for each time point over two experiments. (c) Left panel- The number of cells with Rad52-GFP foci was quantified in Sin3-aid cells prior to and 2 hours after addition of 500 μM IAA. 600 cells were scored for each condition. Right panel- Representative images of chromatin spreads from Sin3-aid cells prior to and 2 hours after addition of 500 μM IAA. (d) The number of cells with Rad52-GFP foci was quantified in Sin3-aid cells not arrested, or arrested with alpha-factor or Nocodazole prior to and 1 hour after addition of 500 μM IAA.
FIGURE 7
FIGURE 7. Deleting RNH1 in transcription mutants synergistically increases YAC and chromosome III instability
(a) RNA biogenesis-RNase H double mutants carrying the YAC were assayed for instability. Error bars represent standard deviation calculated from at least 4 independent colonies. (b) RNA biogenesis and RNase H mutants with a modified chromosome III were assayed for instability. Error bars represent standard deviation calculated from at least 4 independent colonies.
See this image and copyright information in PMC

Comment in

References

    1. Aguilera A, Gómez-González B. Genome instability: a mechanistic view of its causes and consequences. Nature reviews Genetics. 2008;9:204–17. - PubMed
    1. Aguilera A, Klein H. HPR1, a novel yeast gene that prevents intrachromosomal excision recombination, shows carboxy-terminal homology to the Saccharomyces cerevisiae TOP1 gene. Molecular and cellular biology. 1990;10:1439–51. - PMC - PubMed
    1. Alabert C, Bianco J, Pasero P. Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1branch of the S-phase checkpoint. EMBO J. 2009;28:1131–41. - PMC - PubMed
    1. Cerritelli S, Crouch R. Ribonuclease H: the enzymes in eukaryotes. The FEBS journal. 2009;276:1494–505. - PMC - PubMed
    1. Chávez S, Aguilera A. The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genome instability. Genes & Development. 1997;11:3459–70. - PMC - PubMed

Publication types

MeSH terms