Journal: Genes & Development
Article Title: DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase
doi: 10.1101/gad.184697.111
Figure Lengend Snippet: Pfh1 enhances the viability of cells with the RuraR cassette, and its depletion causes fork pausing and converged forks, which are eliminated in swi1Δ cells. (A) Spot assay using fivefold serial dilutions of cells on EMMS or EMMS containing 5 μg/mL thiamine. (B) Growth curves showing population doublings in cultures growing on EMMS or EMMS + 30 μM thiamine (data for nmt-pfh1-GFP+thiamine were also shown in Fig. 1C). (*) P < 0.05 was determined by two-tailed Student's t-test for 48 h of growth in thiamine for all three strains. For 24-h-growth time points, P < 0.05 was determined for RuraR-nmt-pfh1-GFP (**) and RuraR-nmt-pfh1-GFP+thiamine (*), as well as for RuraR-nmt-pfh1-GFP (**) and nmt-pfh1-GFP+thiamine (*). (C, top) Protein samples from RuraR nmt-pfh1-GFP collected after 0, 6, 9, 12, and 24 h in the presence of thiamine and analyzed by immunoblotting with anti-Pfh1 antibody. (Bottom) Coomassie Blue staining of the same membrane. (D, top left) A schematic of the RuraR region on chromosome III. (E) EaeI. (Top right) Panels showing expected replication intermediates during replication of the RuraR cassette are marked with letters a, b, and c. (Bottom left) Cartoon of 2D gel analysis indicates the replication intermediates shown in the panels on top. The fold differences after 6 h of Pfh1 depletion compared with the nondepleted conditions are stated below each letter in the cartoon. (Bottom right) 2D gel analysis of EaeI-digested DNA from RuraR nmt-pfh1-GFP or RuraR nmt-pfh1-GFP swi1Δ cells grown in the presence of thiamine for 0 or 6 h. Digestion by EaeI results in a 6.8-kb fragment. (Arrows) 2N spots. (E) Spot assay using fivefold serial dilutions of cells on EMMS or EMMS+5 μg/mL thiamine. Three independent isolates of nmt-pfh1-GFP swi1Δ cells, in addition to nmt-pfh1-GFP, nmt-pfh1-GFP RuraR swi1Δ, and RuraR swi1Δ cells, were tested.
Article Snippet: We examined replication through these sites in Pfh1-depleted and wild-type cells using two-dimensional (2D) gel analysis, which separates replication intermediates by size in the first dimension and by shape (and size) in the second dimension ( Brewer and Fangman 1987 ).
Techniques: Spot Test, Two Tailed Test, Western Blot, Staining, Membrane, Two-Dimensional Gel Electrophoresis
![Pfh1 <t>facilitates</t> <t>replication</t> fork progression through rDNA in a Swi1-dependent manner. (A) Schematic of the rDNA region used for <t>2D</t> gel analysis (here and elsewhere, schematics are drawn to scale). (X) XhoI; (H) HindIII; (B) BglII; (S) SalI recognition sites. Each repeat contains an origin of replication (ORI3001) (black circle). The RFB region is located between the XhoI and HindII sites. Swi1-dependent (*) and Swi1-independent (**) pause sites are indicated above each RFB. (B) Panels represent different replication intermediates during replication of rDNA. Replication forks migrating leftward from the origin are blocked by the RFB, while replication forks migrating in the same direction as transcription are not affected. (C, left) Cartoon representation of 2D gel analysis for DNA isolated from nmt-pfh1-GFP or nmt-pfh1-GFP swi1Δ cells. Letters on the arc indicate the replication intermediates shown in the panels in B. Here and in other figures: (1N) Nonreplicating DNA fragments; (2N) almost fully replicated DNA fragments; (bu) bubble arc; brackets mark quantified regions outside the pause sites. The BglII (B) SalI (S) fragment used for the 2D gel analysis is 7.4 kb. (Right) 2D gel analysis of nmt-pfh1-GFP or nmt-pfh1-GFP swi1Δ cells grown in the presence of thiamine for 0 or 12 h. Genomic DNA was digested with BglII and SalI. (*) Position of Swi1-dependent RFBs; (**) Swi1-independent RFBs. Numbers indicate RFBs ([1] Ter1; [2] Ter2; [3] Ter3; [4] RFP4). The gels were quantified in ImageQuant, and the numbers in the cartoon represent the fold differences between the 0-h and 12-h thiamine conditions. The fold difference is the average of two independent biological replicates.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5119/pmc03315119/pmc03315119__581fig2.jpg)
![Pfh1's activity is needed at 5S rRNA genes. (A, left) Schematic image of 5S rRNA.03 on chromosome III. Here and in other figures, origins of replication (closed circles) and origin efficiencies (ori eff) are from Heichinger et al. (2006). Here and in other figures, gray boxes indicate hybridization probes. (Right) Panels to the right illustrate replication intermediates formed during replication of 5S rRNA.03. Here and in other figures, lowercase letters mark these intermediates. (B, left) Cartoon of 2D gels analysis of 6-kb DNA EcoRV fragment. Letters indicate replication intermediates in A. (Right) 2D gel analysis from DNA isolated from nmt-pfh1-GFP cells grown in the presence of thiamine for 0 or 12 h. (Arrow) Converged forks. The fold change shown below each letter in the cartoon ([a] 32-fold; [b] threefold; [c] 170-fold) is the average difference between the 0-h and 12-h time points and was calculated from two independent biological replicates. (C, left) Schematic of 5.3-kb AgeI fragment containing the 5S rRNA.37 region from chromosome II. (A) AgeI site. (Right) Schematic showing expected replication intermediates during replication of the 5S rRNA.37 gene. (D, left) Cartoon of 2D gel analysis indicates replication intermediates shown in the panels on the top. (Right) 2D gel analysis of nmt-pfh1-GFP cells grown in the presence of thiamine for 0 or 12 h. Three discrete pause sites were visualized in Pfh1-depleted cells. We suggest that two of these correspond to either paused leftward-moving forks from ORI2128 or paused rightward-moving forks from ORI2127 at the 5S rRNA gene. The third one could be a pause site at the LTR positioned 600 bp downstream from the 5S rRNA gene, almost in the middle of the AgeI fragment. There were 10-fold (a) and threefold (b) increased pausing at the 5S rRNA gene after 12 h of Pfh1 depletion compared with the nondepleted conditions. The level of converged forks was 13-fold higher (c) in Pfhl-depleted cells versus nondepleted cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5119/pmc03315119/pmc03315119__581fig3.jpg)
