Nature. 2011 Aug 7;477(7362):115-9. doi: 10.1038/nature10331.

Protection of repetitive DNA borders from self-induced meiotic instability.

Vader G, Blitzblau HG, Tame MA, Falk JE, Curtin L, Hochwagen A.

Source

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.

Abstract

DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, owing to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination. In the repetitive ribosomal DNA (rDNA) array of the budding yeast Saccharomyces cerevisiae, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin formation. Here we show that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity in the rDNA array. We find that this localized DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination increased specifically in the outermost rDNA repeats, leading to NAHR and rDNA instability. Notably, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2Δ cells. Thus, although the activity of Sir2 globally prevents meiotic DSBs in the rDNA, it creates a highly permissive environment for DSB formation at the junctions between heterochromatin and euchromatin. Heterochromatinized repetitive DNA arrays are abundant in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, the protection of which may be a universal requirement to prevent meiotic genome rearrangements that are associated with genomic diseases and birth defects.

PMID:: 21822291; [PubMed - indexed for MEDLINE]
PMCID:: PMC3166416

Free PMC Article

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Figure 1

rDNA-associated DSB formation and recombination

a, CHEF analysis of the rDNA of meiotic dmc1Δ (H5217), sir2Δ dmc1Δ (H2953), and pch2Δ dmc1Δ (H5216) cells. Schematic denotes the analysed XhoI restriction fragment. A dmc1Δ mutation was used to prevent DSB repair. Mean (+ s.e.m.) of five experiments is shown. Significance (one-tailed Student’s T-test): dmc1Δ vs. sir2Δ dmc1Δ, p-value=0.00122; dmc1Δ vs. pch2Δ dmc1Δ, p-value=0.254; pch2Δ dmc1Δ vs. sir2Δ dmc1Δ, p-value=0.00216. b, Schematic of markers inserted in unique single-copy sequences within 500 bp of the rDNA, and crossover rates in wild-type (H3026; n=467) and pch2Δ (H327; n=186 cells. c, Schematic indicating marker locations in the rDNA used in d and g. URA3 markers were inserted in the NTS1/2 region of indicated repeats. d, Southern blot for restriction fragments containing the indicated rDNA repeat units from pch2Δ dmc1Δ strains #1:H5622, #3:H5636, #10:H5706 (PflMI/SalI; probe: unique rDNA insertion). YCR047C (HindIII) was a positive control for DSB formation. e, Southern blot of left rDNA flank including the outermost rDNA repeat in dmc1Δ (H5583) and pch2Δ dmc1Δ (H5622) cells (SalI/NruI; probe: YLR152C). f, ssDNA enrichment profile of chromosome XII in dmc1Δ (H118, grey), pch2Δ dmc1Δ cells (H2629, black) and sir2Δ dmc1Δ cells (H2953, slate grey). Arrowheads indicate >2-fold elevated DSB formation in pch2Δ dmc1Δ compared to dmc1Δ. g, Tetrad analysis of pch2Δ URA3 rDNA insertions strains (#1:H4611, #3:H4613, #10:H3823, #12:H4612, #29:H3820, #49:H3821; Table S2). Recombination rates between URA3 and rDNA-flanking markers are shown in relation to the physical URA3 positions within the rDNA. h, Relative contribution of each measured interval indicated in g to total rDNA recombination (percent of crossovers/kb of interval). i, Incidence of changes in rDNA repeat number between URA3 insertion and the rDNA boundary in pch2Δ tetrads that had undergone crossover recombination. j, CHEF analysis of two tetrads that have undergone unequal recombination (rec.) with parental controls (par.; XhoI; probe: NTS1).

Protection of repetitive DNA borders from self-induced meiotic instability

Nature. 2011 August 7;477(7362):115-119.