Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements - PubMed (original) (raw)

Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic loci containing pausing elements

Daniele Fachinetti et al. Mol Cell. 2010.

Abstract

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.

Copyright (c) 2010 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Replication fork dynamics and fusion

wt (sy2201) cells were arrested in G1 and released in S-phase with BrdU in 3 different sets of experiments (untreated 16°C, HU and HU recovery). Orange histogram bars (BrdU) in the Y-axis represent the average signal ratio of loci significantly enriched in the immunoprecipitated fraction (IP) along the indicated regions in log2 scale (detection p-value and change p-value are <0.001. Light orange bars should contain at least 10 contiguous probes with a p-value <0.001). The X-axis shows chromosomal coordinates. ARS elements are indicated (red lines) and the blue bars mark the ORFs. (A) Examples of fork movement monitored by BrdU incorporation at ARS305 with method 1, 2 and 3. See Table S2 for the list of the origin-related BrdU peaks (B) Visualization of 3 termination regions using the 3 methods. Red bars indicate TERs. Replication origins and experimental conditions are shown. The green line indicates the centromere. See Figure S1 and Table S3 for TERs size and position.

Figure 2

Figure 2. Identification of pausing elements at TERs

TER1202 (CHRXII), TER1102 (CHRXI) and TER1005 (CHRX) are shown as examples. BrdU-labeled forks are indicated in orange color and derive from the analysis carried out in strain sy2201 using the conditions for experiments 1 (TER1102) or experiment 2 and 3 (TER1202 and 1005) respectively. In each panel, the top part shows the extension of fork movements (black arrows) at the indicated time points based on BrdU data. In each panel, the bottom part represents a selected time point when forks reach the TER area. The black circle within TER1202 indicates the centromere. The green peaks in the bottom part of the TER1102 panel indicate the S-phase clusters of the Pol III subunit Rpc25 using strain cy8735. The blue peaks in the bottom part of the TER1005 panel indicate the S-phase clusters of the Pol II subunit Rpb3 using strain cy8519 (see methods section for details). Red arrows indicate transcription direction. Red bars mark the TER zones. The Venn diagram shows the relative number of TERs containing centromere (orange), Pol II (blue) and Pol III (green). See also Tables S4 for list of TERs containing pausing elements.

Figure 3

Figure 3. Rrm3 contributes to fork progression across TERs

(A-C) wt (sy2209) and rrm3Δ (cy6807) cells were pre-synchronized in G2 with Nocodazole and released in α-Factor. Cells were then released in S-phase at 23°C and samples collected at 40′. Genomic DNA was analyzed by 2D gels. Schematic representations of the different fork pausing and termination signals are shown. The red letters in panel b indicate double Y and Xs respectively. The red numbers indicate pausing sites (see text for details). Relative BrdU maps, restriction digestion strategy and 2D gels quantification are shown in Figure S3.

Figure 4

Figure 4. Top2 is required for efficient replication termination

The ChIP-chip data are described as in Figure 1. (A) BrdU-labeled forks are indicated in orange. Top2-10Flag (cy7315) cells were arrested in G1 with α-factor and released at 23°C in the presence of HU for 1 hour or in the presence of nocodazole for 3 hours. Samples were collected at the indicated time points and processed for ChIP-chip analysis. As a control we show the BrdU-maps (in orange) that correspond to forks that have experienced 1h in HU. Green histogram bars represent the Top2 clusters in HU (IP-Top2 HU) and the blue ones indicate the Top2 clusters in nocodazole (IP-Top2 N). Red bars indicate the TER zones. (B) wt (sy2201) and top2-1 (cy7421) cells were released from α-factor in YPD with BrdU and HU at 37°C for 1 hour. BrdU maps of wt cells experiencing HU treatment at 23°C is also shown. Red bars indicate the TER zones. List of TERs containing Top2 clusters are shown in table S4 and the relative statistical analysis in table S5.

Figure 5

Figure 5. Top2 is required for chromosome resolution

(A-B) wt (cy7627), top2-1 (cy7671) and top3Y356F (cy 7629) cells were arrested in G1 with α-Factor and released in S-phase in YP+ Gal at 37°C in the presence of Nocodazole. Genomic DNA was extracted in agarose plugs at the indicated time points. Yeast chromosomes were separated by PFGE and analyzed by Southern blotting with the TER302 probe. M indicates the chromosome marker. DSBs indicate double strand breaks. (B) Agarose plugs were digested with _Eag_I. Schematic representation of the analyzed region is shown. (C) wt (cy7627), top2-1 (cy7671) and top3Y356F (cy 7629) were released in S-phase at 37°C and different samples (30′-40′-50′) were pulled together to increase the chance to visualized the replication intermediates. 30 μg of DNA were digested with _Hind_III and _Pst_I and analyzed by 2D gels using TER302 probes. FACS, PFGE and 2D gels are also shown in Figure S4.

Figure 6

Figure 6. Top2 prevents fragility at TERs

(A) top2-1 (cy8423) cells were released from G1 in S phase at 37°C. The sample was collected after 150 min (following cell division) and processed for ChIP-chip with antibodies against γH2A. The red histogram bars represent the γH2A clusters. BrdU-labeled forks (orange, IP-BrdU) and Top2 peaks (green, IP-Top2 HU) obtained from independent experiments are also shown. The red bars mark the TERs. See also Table S4 (B) top2-1 (cy7671) cells were released from G1 in S-phase at 25°C or 37°C in the presence of nocodazole to compare the relative genomes within one cell cycle. Samples were collected after 2,5 hour and processed for CGH analysis. The plot of the log2 ratio value on Y-axis shows DNA copy number changes between test-DNA and reference-DNA. The different colors represent all 16 chromosomes and the corresponding number is indicated. (C) SignalMap ver1.9 (NimbleGen) magnification of 4 regions detected by CGH. Plot of the log2 ratio value on Y-axis shows DNA copy number changes. The X-axis shows chromosomal coordinates. Black arrows indicate sites of genomic instability. Red bars indicate position of TER sites. Genome instability regions are also shown in Figure S1 and Table S6.

Figure 7

Figure 7. A model for replication termination

I) Precatenane resolution: Top2 mediates fork progression at the TER zone by resolving precatenanes behind the forks. II) Fork fusion: the right fork stalls at a pausing site (pausing element, red symbol) and emerges with an asymmetric conformation. The leading polymerase (black oval) and the lagging apparatus (yellow oval) are shown. III) Catenation: Top2 then resolves the last catenation at TERs before DNA segregation allowing chromosomes resolution.

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References

    1. Admire A, Shanks L, Danzl N, Wang M, Weier U, Stevens W, Hunt E, Weinert T. Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast. Genes Dev. 2006;20:159–173. - PMC - PubMed
    1. Azvolinsky A, Dunaway S, Torres JZ, Bessler JB, Zakian VA. The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes. Genes Dev. 2006;20:3104–3116. - PMC - PubMed
    1. Azvolinsky A, Giresi PG, Lieb JD, Zakian VA. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae. Mol Cell. 2009;34:722–734. - PMC - PubMed
    1. Bachant J, Alcasabas A, Blat Y, Kleckner N, Elledge SJ. The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-1 modification of DNA topoisomerase II. Mol Cell. 2002;9:1169–1182. - PubMed
    1. Baxter J, Diffley JF. Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast. Mol Cell. 2008;30:790–802. - PubMed

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