A topoisomerase II-dependent mechanism for resetting replicons at the S-M-phase transition - PubMed (original) (raw)
A topoisomerase II-dependent mechanism for resetting replicons at the S-M-phase transition
Olivier Cuvier et al. Genes Dev. 2008.
Abstract
Topoisomerase II (topo II) is required for chromosome segregation and for reprogramming replicons. Here, we show that topo II couples DNA replication termination with the clearing of replication complexes for resetting replicons at mitosis. Topo II inhibition impairs completion of DNA replication, accounting for replication protein A (RPA) stabilization onto ssDNA. Topo II inhibition does not affect the caffeine-sensitive ORC1 degradation found upon origin firing, but it impairs the cdk-dependent degradation/chromatin dissociation of an ORC1/2 reservoir at mitosis. Our results show that ORC1 degradation is rescued by Pin1 depletion and that this topo II-dependent clearing of ORC1/2 from chromatin involves the APC.
Figures
Figure 1.
Topo II is required for the dissociation from chromatin of ORC and RPA. (A) Interphase egg extracts were immunodepleted with anti-topo II antibodies and loaded (lanes 1,2) in parallel with a dilution series of mock-depleted extracts (1×, 0.2×, or 0.04×; lanes 3–5). Immunoblotting analyses were performed with the indicated antibodies. (B) Sperm chromatin was incubated with interphase topo II- or mock-depleted egg extracts before inducing mitosis. Chromatin was purified and stained with anti-biodUTP (panels b,e,h,k) or anti-ORC2 (panels c,f,i,l). (Panels a,d,g,j) DNA was counterstained with Hoechst 33258. Bar, 10 μm. (C) Four-fifths of the reaction in B was used to analyze chromatin-associated proteins by immunoblotting. (D) Confocal microscopy of mitotic ORC and RPA foci in topo II-depleted chromatin as described in B. Chromatin was stained with anti-RPA (panel a) and anti-ORC2 (panel b). Panel c shows the merged image with enlarged section (3×). Bar, 5 μm.
Figure 2.
Topo II is critical for ORC/RPA removal after initiation of DNA replication. (A) Scheme of the experiments. The dissociation of RPA and ORC from chromatin was measured after replicating chromatin in topo II-depleted extracts (protocol 1), or without prior initiation of DNA replication (protocol 2). (B) Sperm chromatin was incubated with topo II-depleted S-phase egg extracts to allow DNA replication to proceed for 120 min. Fresh S-phase egg extracts were added in the absence (panels a–d) or in the presence (panels e–h) of aphidicolin for an additional 30 min, and a mitotic extract was added to induce mitosis. As an additional control, the same amounts of fresh mitotic egg extracts were added after only 15 min. In this condition, mitosis is induced without initiation of DNA replication (Cuvier et al. 2006). The topo II-specific inhibitor ICRF was also added to further inhibit topo II activity at mitosis. Chromatin was purified and stained with the indicated antibodies. (Panels a,e,i,m) DNA was counterstained with Hoechst. Bar, 10 μm.
Figure 3.
ICRF blocks the complete degradation of ORC1 at mitosis. (A) Sperm chromatin was incubated with S-phase egg extracts to allow DNA replication to proceed for 120 min in the presence or absence of ICRF (added at t45). Chromatin-associated protein samples were analyzed by immunoblotting with anti-ORC1/2 or anti-RPA32/14 antibodies and nucleoplasmic fractions with anti-phosphorylated chk1 (chk1-P) or anti-H3 antibodies. (B) Sperm chromatin was assembled in extract after treatments with ICRF as in A and/or aphidicolin added after 1 h and analyzed by immunoblotting. (C) Sperm chromatin was incubated with S-phase egg extracts to allow DNA replication to proceed for 120 min in the presence or absence of ICRF (t45) or aphidicolin (t60) as indicated. Chromatin-associated proteins (bound) were purified through a sucrose cushion (left panels) after detergent extraction of soluble proteins (chromatin unbound; right panels). Samples were analyzed by immunoblotting with the indicated antibodies. The arrowhead indicates a degradation product of ORC1. (D) Same as described in C, except that cyclin B was added after purification of interphase chromatin to drive mitosis for 120 min. Chromatin-associated proteins (bound) were purified after detergent extraction of soluble proteins (unbound) as in C. The arrowhead indicates a degraded form of ORC1 that is only seen at mitosis.
Figure 4.
The topo II-dependent block of ORC1 degradation at mitosis is caffeine-insensitive but cdk- and Pin1-dependent. (A) Experimental scheme of the experiment shown in B. (B) Sperm chromatin was incubated with S-phase egg extracts for 120 min in the presence or absence of ICRF added in S phase (t45min; lane 3) or 15 min before (t90min, lanes 4–6) or after (t135min, lane 7) mitotic entry. (Lanes 1,2) Aphidicolin was added at 45 min, and caffeine was added at the time indicated, before or after addition of cyclin B (t120) to drive mitosis. Chromatin-associated proteins were purified at mitosis and analyzed by immunoblotting. (C) Interphase egg extracts were mock-depleted (left) or immunodepleted with anti-Pin1 antibodies (right) and analyzed with the indicated antibodies. (D) Immunodepletion of Pin1 impairs ORC1 stability in ICRF-treated extracts. Sperm chromatin was incubated with Pin1-depleted or mock-depleted S-phase extracts and driven into mitosis as described in B. Chromatin-associated proteins were analyzed by immunoblotting.
Figure 5.
Inhibition of Topo II activity prevents the completion of DNA replication. (A) Sperm chromatin was incubated in a Xenopus egg extract and 20 μM ICRF was added either at time 0 or after 45 min with or without caffeine. DNA synthesis was measured by the incorporation of 32PdATP (Materials and Methods). Percentage replication relative to input sperm chromatin is indicated. (B) Sperm chromatin was incubated with egg extracts treated with ICRF or a buffer control. The incorporation of BrdU (green) was monitored after 1 h for visualization of actively replicating regions (r). The graph shows the proportion of actively versus inactively replicating regions marked by “gaps” (g; >15 kb), measured along >10 Mb of combed DNA. (C) Same as in B, except that a second nucleotide analog (DIG-dUTP, in red) was added after 1 h of incubation to mark the progression of forks. The graph shows the proportion of symmetric forks (Sym) versus asymmetric forks (Asym), which harbor DIG-dUTP at only one side of the BrdU track. (D) Same as in C, except that BrdU was present during the whole S phase (2 h; ±ICRF) and DIG-dUTP (red) was added together with a fresh egg extract, after removal of ICRF, to mark unreplicated sites. The graph shows the proportion (along >13 Mb of combed DNAs) of terminated (T) or nonterminated (nt) replicons, where incorporation of the second nucleotide was visible. Note: Each dot represents ∼2 kb of unreplicated DNA (see Supplemental Figs. S8, S9).
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