Article Title: The mechanism of DNA replication termination in vertebrates
Figure Lengend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
Article Snippet: To analyze topoisomers ( ) 0.25 ng/µl of radiolabelled DNA was incubated in 1X Buffer A and 1X Buffer B (Topogen) with 0.2 U/µl Human Topo II-α (Topogen) at 37°C for 15 minutes, or in CutSmart Buffer with 0.4 U/µl XmnI or 0.04 U/µl Nt.BspQI (New England Biolabs) for 1 hour.
Techniques: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation