Journal: Cell reports

Article Title: Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination

doi: 10.1016/j.celrep.2019.08.097

Figure Lengend Snippet: (A) Schematic of the decatenation assay. (B) Top2α-immunodepleted extracts were visualized alongside mock-immunodepleted extracts by western blotting. The dark panel is overexposed. (C) Plasmid DNA was replicated in mock-, Top2α-immunodepleted, and rescued (ΔTop2α+hTop2α) extracts containing [α− 32 P]dATP. Replication intermediates were purified and then separated on an agarose gel and visualized by autoradiography. See also – and . (D) Quantification of decatenation in (C). See also . (E) 500 ng of human Top2α was separated on a polyacrylamide gel and visualized by Coomassie staining. (F) DNA synthesis was measured during plasmid DNA replication in mock- and Top2α-immunodepleted extracts. Plot depicts mean ± SD from 4 experiments. (G) Schematic of the fork merger assay. (H) DNA samples from (E) were digested and then separated on an agarose gel and visualized by autoradiography. See also . (I) Quantification of fork merger in (H). See also .

Article Snippet: [α− 32 P]dATP , Perkin Elmer , BLU512Z250UC.

Techniques: Western Blot, Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Autoradiography, Staining, DNA Synthesis

Journal: Cell reports

Article Title: Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination

doi: 10.1016/j.celrep.2019.08.097

Figure Lengend Snippet: (A) Schematic of the fork merger assay. (B) Plasmid DNA replication was performed in mock- or Top2α-immunodepleted extracts containing [α− 32 P]dATP and either vehicle or Top2-i. Replication intermediates were purified, digested, and then separated on an agarose gel and visualized by autoradiography. See also . (C) Quantification of fork merger in (B). See also . (D) Schematic of the decatenation assay. (E) Replication intermediates from (B) were separated on an agarose gel and visualized by autoradiography. See also . (F) Quantification of Cats− and Cats+ in (E). See also – . (G) Schematic of DNA structures observed by 2D gel analysis of nicked replication intermediates. The source of the θs, Cats, and cm is indicated in (D). Linear monomers (LMs) arise from a double-strand breaks in cm, while σs arise from double-strand breaks in θs. Each catenated species runs at a discrete location corresponding to the number of catenanes present. Numbers indicate how many catenated linkages are present. (H) Plasmid DNA was replicated in Top2α-immunodepleted extracts containing [α− 32 P]dATP and either Vehicle or Top2-i. Replication intermediates were purified, nicked, and then separated by 2D gel electrophoresis and detected by autoradiography. See also . (I and J) Quantification of the number of catenanes present in the catenanted species detected in (Hii), (Hiii), (Hv), and (Hvi). The graphs depict cumulative frequency distribution, and numbers in parentheses indicate the median number of catenanes in each condition. See also – .

Article Snippet: [α− 32 P]dATP , Perkin Elmer , BLU512Z250UC.

Techniques: Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Autoradiography, Two-Dimensional Gel Electrophoresis, Electrophoresis

Journal: Cell reports

Article Title: Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination

doi: 10.1016/j.celrep.2019.08.097

Figure Lengend Snippet: (A) Schematic of DNA structures observed by 2D gel electrophoresis following restriction digest (as in ). Double Ys and Xs arise from intermediates cut behind two forks, while bubbles occur when intermediates are cut between two forks. Ys arise from incision of one fork (see ). (B) Plasmid DNA was replicated in mock- and Top2α-immunodepleted extracts containing [α− 32 P] dATP. To monitor the DNA structures formed, replication intermediates were digested and then separated by 2D gel electrophoresis and detected by autoradiography. See also . (C) Plasmid DNA was replicated in Mock- and Top2α-immunodepleted extracts containing [α− 32 P] dATP. Replication intermediates were purified then denatured. The expected products are shown. Black lines indicate unlabeled parental strands. Dashed lines represent strands that should not be detectable because they are unlabeled or not intertwined with a labeled strand. (D) Schematic depicting the effect of Nb.BssSI treatment on the intermediates shown in (C). (E) Replication intermediates from (C) and (D) were separated on a denaturing agarose gel and visualized by autoradiography. Structures indicated in (C) and (D) are labeled. Linear (3,402) molecules arise from double-stranded monomers that were nicked during DNA purification, prior to Nb.BssSI treatment. Lanes 10–14 are molecular weight standards of the indicated sizes. The numbers in parentheses represent the amount of unreplicated DNA in lanes 2–9 and 15–22 that each standard corresponds to. See also and – . (F) Quantification of the amount of unreplicated DNA in lane 6 of (E) after correcting for the ~60-bp footprint of the replisome . Plot depicts mean ± SD from 4 experiments. (G) Plasmid DNA containing a LacR-bound 16x lacO array was replicated in mock- and Top2α-immunodepleted extracts containing [α− 32 P]dATP. Replication intermediates were purified and then nicked and denatured. (H) Replication intermediates from (G) were separated on a denaturing agarose gel alongside a DNA ladder. Note that lanes 1 and 10 are overexposed. See also – . (I) Quantification of the size of the nascent strand products from (H) relative to the initial size of the nascent products in mock-immunodepleted extracts. Plot depicts mean ± SD from 3 experiments. (J) Quantification of the abundance of the leading strand (~948) products from (H) relative to the initial abundance in mock-immunodepleted extracts. Plot depicts mean ± SD from 3 experiments.

Article Snippet: [α− 32 P]dATP , Perkin Elmer , BLU512Z250UC.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Autoradiography, Purification, Labeling, Agarose Gel Electrophoresis, DNA Purification, Molecular Weight

Journal: Cell reports

Article Title: Topoisomerase II Is Crucial for Fork Convergence during Vertebrate Replication Termination

doi: 10.1016/j.celrep.2019.08.097

Figure Lengend Snippet: (A) Schematic of the ligation assay. Dashed lines represent unlabeled parental strands, which are not detected. (B) Plasmid DNA was replicated in mock-, Top2α-immunodepleted, and rescued (ΔTop2α+hTop2α) extracts containing [α− 32 P]dATP. Replication intermediates were purified, digested, and then separated on a denaturing agarose gel and visualized by autoradiography. See also . (C) Quantification of ligation in (B). See also . (D) Fork merger and ligation were quantified from multiple experiments. Plot depicts mean ± SD from 4 experiments. See also . (E) Plasmid DNA replication was performed in mock- and Top2α-immunodepleted extracts, and chromatin-bound proteins were recovered at different time points by plasmid pull-down. Approximate kinetics of DNA replication are indicated. (F) Chromatin associated proteins from (E) were detected by western blotting. (G–J) Binding of MCM6 (G), CDC45 (H), RPA70 (I), and RPA30 (J) to chromatin was quantified from (F). See also – .

Article Snippet: [α− 32 P]dATP , Perkin Elmer , BLU512Z250UC.

Techniques: Ligation, Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Autoradiography, Western Blot, Binding Assay

Journal: Molecular Cell

Article Title: Repression of Divergent Noncoding Transcription by a Sequence-Specific Transcription Factor

doi: 10.1016/j.molcel.2018.10.018

Figure Lengend Snippet:

Article Snippet: dATP [α-32P] , PerkinElmer , NEG512H500UC.

Techniques: Recombinant, Hybridization, Labeling, Concentration Assay, Reverse Transcription, SYBR Green Assay, Western Blot, RNA Sequencing, Software