Transcription-dependent recombination and the role of fork collision in yeast rDNA - PubMed (original) (raw)

. 2003 Jun 15;17(12):1497-506.

doi: 10.1101/gad.1085403. Epub 2003 Jun 3.

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Transcription-dependent recombination and the role of fork collision in yeast rDNA

Yasushi Takeuchi et al. Genes Dev. 2003.

Abstract

It is speculated that the function of the replication fork barrier (RFB) site is to avoid collision between the 35S rDNA transcription machinery and the DNA replication fork, because the RFB site is located near the 3'-end of the gene and inhibits progression of the replication fork moving in the opposite direction to the transcription machinery. However, the collision has never been observed in a blockless (fob1) mutant with 150 copies of rDNA. The gene FOB1 was shown previously to be required for replication fork blocking activity at the RFB site, and also for the rDNA copy number variation through unequal sister-chromatid recombination. This study documents the detection of fork collision in an fob1 derivative with reduced rDNA copy number (approximately 20) using two-dimensional agarose gel electrophoresis. This suggests that most of these reduced copies are actively transcribed. The collision was dependent on the transcription by RNA polymerase I. In addition, the transcription stimulated rDNA copy number variation, and the production of the extrachromosomal rDNA circles (ERCs), whose accumulation is thought to be a cause of aging. These results suggest that such a transcription-dependent fork collision induces recombination, and may function as a general recombination trigger for multiplication of highly transcribed single-copy genes.

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Figures

Figure 1.

Figure 1.

(A) Structure of rDNA repeats in Saccharomyces cerevisiae. The locations of the 35S and 5S rRNA genes (the direction of transcription is indicated by arrows) are shown in the upper part. The recognition sites of _Bgl_II are also shown. The two nontranscribed spacer regions (NTS1 and NTS2), the ARS (replication origin), and its surrounding regions are expanded below. The RFB (replication fork barrier site) is indicated. The striped box is the region used for the rDNA-specific probe. The solid bars I and E are the components of HOT1. Two arrows in the bottom part show progression of one of the replication forks started from the ARS, bidirectionally. The upper fork (WT, wild type) is arrested at the RFB, and the lower one (TAK300; fob1, low rDNA copy number) is slowed down in the 35S rDNA. SDR is the region in which the replication fork is slowed down (Fig. 2C, panel c). The recognition sites of _Sph_I and _Bgl_II, which were used for the 2D gel analysis (Fig. 2C), are also shown. (B) Structure of the helper plasmid, pRDN-hyg1 (Chernoff et al. 1994). The filled box shows an rDNA unit. The hygromycin B-resistance mutation site is indicated in the 18S rDNA. This plasmid was used to isolate strains with low copy numbers of rDNA repeats.

Figure 2.

Figure 2.

Analysis of low-copy-rDNA strains. (A) Southern hybridization analysis of rDNA copy numbers. DNA was digested with _Bgl_II and subjected to electrophoresis followed by Southern analysis using the rDNA probe (Fig. 1). A single-copy gene, MCM2, was used as an internal control for normalization. (B) Quantitation of the intensities of the bands. NOY408-1b (wild-type strain), NOY408-1bf (fob1), TAK300 (fob1; low-copy rDNA strain), TAK301 (fob1 pol1; low-copy rDNA strain). (C) Collision between the transcription and the replication machineries analyzed by 2D gel analysis. DNA was prepared from the strains indicated, digested with _Bgl_II and _Sph_I, and subjected to 2D agarose gel electrophoresis followed by Southern hybridization using the rDNA probe (see Fig. 1). A spot indicated by an arrowhead shows accumulation of Y-shaped DNA molecules at the RFB site (panel a). The slowdown region (SDR) is located between two arrows (panel c). The numbers in parentheses are copy numbers of rDNA in each strain. (Panel a) NOY408-1b (wild-type strain). (Panel b) NOY408-1bf (fob1). (Panel c) TAK300 (fob1; low-copy rDNA strain). (Panel d) TAK301 (fob1 pol1; low-copy rDNA strain). (Panel e) TAK301, complemented by a plasmid-borne RPA135 gene (fob1 POLI; low-copy rDNA strain).

Figure 3.

Figure 3.

Detection of the extrachromosomal rDNA circles (ERCs). DNA isolated from various strains was subjected to electrophoresis (0.6% agarose gel for 20 h at 1 V/cm) followed by Southern hybridization using the rDNA probe (see Fig. 1). The positions of monomer ERC, dimer ERC, and genomic rDNA are indicated by arrows. The strains are the same as used in Figure 2C. We used the same number of cells, which were determined by spectrometer to isolate the DNA.

Figure 4.

Figure 4.

Analysis of rDNA repeat size by pulsed-field gel electrophoresis (PFGE; CHEF, Bio-Rad). DNA was isolated using agarose gel blocks, digested with _Bam_HI, and subjected to PFGE, followed by hybridization with the rDNA probe (see Fig. 1). As there is no _Bam_HI recognition site in the repeats, the length of the fragment indicates the rDNA copy number. This fragment includes 39 kb of non-rDNA region. The numbers above the autoradiograph show the number of generations after purification from single colonies. (A) TAK300 (fob1; low-copy rDNA strain). (B) TAK301 (fob1 pol1; low-copy rDNA strain). (C) TAK301, complemented by a plasmid-borne RPA135 gene (fob1 POLI; low-copy rDNA strain).

Figure 5.

Figure 5.

Life span analysis. The analysis was performed on NOY408-1b (white box), NOY408-1bf (white circle), and TAK300 (filled circle) as described previously (Kennedy et al. 1994). Life span was determined by scoring the number of daughter cells produced by each mother cell before cessation of all division. The average life spans were as follows: NOY408-1b = 18; NOY408-1bf = 28; TAK300 = 22.

Figure 6.

Figure 6.

The transcription-dependent recombination model for rDNA repeat expansion, contraction, and production of ERCs in TAK300 (low rDNA copy number, fob1). The positions of the ARS and the 35S rDNA are shown as filled dots and arrows, respectively. Individual lines represent chromatids with double-stranded DNA. In this model, DNA replication starts from one of the _ARS_s (ARS-2) bidirectionally (A). As all of the 35S rDNA units are transcribed fiercely in this strain, the rightward replication fork is slowed down in the 35S rDNA (SDR; B), and we propose that it stimulates a DNA double-strand break (indicated by an arrowhead in C). As the strain is fob1, the replication fork barrier (RFB) site at the 3′-end of the 35S rDNA is not functioning. (D) A strand invasion at a homologous duplex (a downstream sister chromatid near ARS-1 in this example) takes place, and a new replication fork is formed. (E) The new replication fork meets with the leftward replication fork, resulting in formation of two sister chromatids, one of which gains an extra copy of rDNA, indicated as a striped line. If the strand invasion is at a site in an upstream repeat (e.g., right side of ARS-3), a loss, rather than a gain, of an rDNA repeat is expected. If the strand invasion is at a site in the same chromatid (_D_′), an ERC is produced (_E_′). Then another invasion takes place (_E_′, lower part) and two sister chromatids are formed (_F_′). The region that is replicated twice is indicated as a striped line in _F_′. A similar model that does not involve transcription-related ERC production is presented by Rothstein and Gangloff (1999).

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