Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation

Abstract

DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600-base-pair (bp) region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M-G1 transition. top1 top2 double mutants exhibit fork block and processing and phosphorylation of Rad53 and gamma H2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation, and chromosome breakage during segregation.

Figure 1.

Top1 and Top2 localization at replicating chromosome III. (A) Schematic representation of the topological problems generated by advancing replication forks: Positive supercoiling [(+)Sc] at the unreplicated portions of a topological domain is generated by fork advance; precatenane nodes, at the replicated duplex, arise as a consequence of fork rotation. (B) Top1-3xFlag (CY6838), Top2-3xFlag (CY6839), and BrdU-incorporating (SY2201) cells were released from α-factor-induced G1 block, treated with 0.2 M HU for 1 h, and processed for ChIP with antibodies specific to the Flag epitopes or BrdU-substituted DNA. Black and yellow histogram bars in the Y-axis show the average signal ratio of loci significantly enriched in the immunoprecipitated fraction along chromosome III in log₂ scale. Black bars correspond to a region represented at higher resolution in the oligonucleotide array, and the histograms are therefore more compacted. The X-axis shows kilobase units. Positions of ARS elements acting as efficient early-firing replication origins on the chromosome are indicated. Red dots correspond to ssDNA-accumulating regions in HU-treated cells (Feng et al. 2006). Blue dots mark the position of potential origins of replication as defined by ORC and MCM protein binding (Wyrick et al. 2001). (C) Magnification of a ∼70-kb region close to the right telomere of chromosome III showing non-origin-related Top2 binding. The horizontal bars indicate ORFs.

Figure 2.

Top1 and Top2 binding at replicating regions is dependent on efficient origin firing. Top1-10xFlag (A) and Top2-10xFlag (B) cells bearing a wild-type (wt) chromosome VI (CY7179 and CY7315, respectively), disrupted ARS607 (CY7363 and CY6379), or mutations in all early origins (shaded in blue) (CY7366 and CY7372) were released from α-factor-induced G1 block, treated with 0.2 M HU for 1 h, and processed for ChIP with antibodies specific to the Flag epitopes. Blue histogram bars in the Y-axis show the average signal ratio of loci significantly enriched in the immunoprecipitated fraction along chromosome VI in log₂ scale. The X-axis shows kilobase units. Positions of all ARS elements acting as replication origins on the chromosome and the CEN sequence are indicated. Blue asterisks indicate the mutated ARSs.

Figure 3.

Top1 and Top2 colocalize with Dbp3 at replication forks. Top1-10xFlag/Dpb3-3xHA (A) and Top2-10xFlag/Dpb3-3xHA (B) cells (CY7340 and CY7343 strains, respectively) were released from α-factor-induced G1 block and treated with 0.2 M HU for 2 h; cultures were then split and processed for ChIP with antibodies specific to either Flag or HA epitopes. Blue histogram bars in the Y-axis show the average signal ratio of loci significantly enriched in the immunoprecipitated fraction along chromosome VI in log₂ scale. The X-axis shows kilobase units. Positions of all ARS sequences acting as replication origins on the chromosome and CEN sequence are indicated. The bottom panels show Top1/Top2 (after 1- and 2-h HU treatment) and Dpb3 (after 2-h HU treatment) clusters surrounding ARS607 element in detail. Blue horizontal bars represent ORFs. Asterisks indicate the position of tRNA genes.

Figure 4.

Effect of Top1 and/or Top2 activity attenuation on chromosomal replication and checkpoint activation. (A) Wild-type (SY359), top1Δ (CY2279), top2-1 (SY2183), and top1 Δtop2-1 (CY7039) cells were arrested in G1 by α-factor treatment at permissive temperature and then released into fresh medium at 37°C. Samples were collected at the indicated time points for FACS analysis and TCA protein precipitation and further immunodetection of Rad53 protein using antibodies recognizing the protein backbone (EL7) or the phosphorylated epitopes only (F9). (B) Wild-type, top1Δ, top2-1, and top1Δ top2-1 cells were arrested in G1 and released into fresh medium containing 0.2 M HU at 37°C. Samples were collected after 2 h, genomic DNA was extracted, and replication intermediates (RI) were analyzed by 2D gel electrophoresis using a probe specific to ARS305 region. (C) Wild-type, top1Δ, top2-1, and top1Δ top2-1 cells were arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points for FACS analysis, TCA protein precipitation, and immunodetection of Rad53 and S129-phosphorylated H2A proteins.

Figure 5.

Aberrant replication intermediates accumulate in top1 top2 mutants. (A) Wild-type (SY359) and top1Δtop2-1 (CY7039) cells were arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points, and genomic DNA was extracted. Replication intermediates were analyzed by 2D gel electrophoresis with ARS305 probe. Black and white arrowheads indicate bubble and small-Y arcs, respectively, in arrested Δtop1 top2-1 cells. TCA protein precipitation of the same samples and immunodetection of Rad53 protein with EL7 antibodies are shown. (B) top1Δ top2-1 (CY7039) and top1Δ top2-1 exo1Δ (CY8002) cells where arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points, and genomic DNA was extracted. Replication intermediates were analyzed by 2D gel electrophoresis with ARS305 probe. A schematic representation of replication intermediates (RIs) and small Ys arising at ARS305 region is shown. (C) top1Δ top2-1 and top1Δ top2-1 exo1Δ cells were arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points for FACS analysis, TCA protein precipitation, and immunodetection of Rad53 with EL7 and F9 antibodies. Quantization of the abundance of Rad53-phosphorylated species detected by F9 antibody immunoblot is shown.

Figure 6.

Lack of Top1 and Top2 activity after replication onset induces persistent Rad9-dependent DNA damage checkpoint activation. (A) top1Δ top2-1 (CY7039), top1Δ top2-1rad9Δ (CY7642), and top1Δ top2-1mrc1Δ (CY7701) cells were arrested in G1, released into fresh medium for 30 min at 25°C, and then transferred to 37°C prewarmed medium. Samples were collected at the indicated time points for FACS analysis, TCA protein precipitation, and immunodetection of Rad53 protein. (B) top1Δ top2-1, top1Δ top2-1 rad9Δ, and Δtop1 top2-1 rad53-K227A (CY7295) cells were arrested in G1, released into fresh medium for 30 min at 25°C, then transferred for 90 min to 37°C prewarmed medium and subsequently back into fresh medium at permissive temperature. Samples were collected at the indicated time points for FACS analysis, TCA protein precipitation, and immunodetection of Rad53 protein.

Figure 7.

Hypothetical models to explain the chromosomal abnormalities that arise in top2 and top1 top2 mutants. (A) Schematic representation of the phenotypes observed in top2-1 mutants. Precatenate formation during initiation and/or termination of DNA replication would require Top2 activity for resolution. Although other possibilities can be envisaged, it is possible that, in the absence of a fully functional Top2 activity, unscheduled strand passage reactions, perhaps mediated by type I DNA topoisomerases, may lead to the generation of sister chromatid interlocking during S phase. Upon anaphase onset, the mechanical tension generated by the mitotic spindle (dashed arrows) might force the separation of the entangled chromatids, leading to the formation of DNA breaks and DNA damage checkpoint signals. (B) Schematic representation of the phenotypes observed in top1Δ top2-1 mutants. The topological constrains arising in the double mutants cause the block of fork progression. Stalled forks could either suffer DNA breaks, perhaps as a consequence of fork collapse at nicks, or resection of nascent chains. Exo1 is likely implicated in the resection of nascent chains (Cotta-Ramusino et al. 2005) and perhaps also in DSB resection together with Mre11 (Nakada et al. 2004). In both cases, RPA–ssDNA filaments could form, leading to checkpoint activation.