Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains - PubMed (original) (raw)
Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains
A Lengronne et al. Nucleic Acids Res. 2001.
Abstract
Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.
Figures
Figure 1
Incorporation of BrdU into chromosomes of _GPD_–_TK_7× yeast cells. (A) Exponential cultures of E001 (wild-type) and E1000 (TK+) cells were grown for 2 h at 25°C in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 400 µg/ml BrdU. Chromosomal DNA samples were prepared and separated by PFGE as described in Materials and Methods (except that the 60 s pulses were for 8 h only) and stained with ethidium bromide. The position of molecular weight markers is shown on the left. Arrows point to chromosome V, 576 and 611 kb (due to the insertion of seven GPD_–_TK copies) in the wild-type and TK+ strains, respectively. (B) The gel shown in (A) was transferred to a nitrocellulose membrane and BrdU was detected using a monoclonal antibody against BrdU and a fluorescently labeled secondary antibody. After FluorImager scanning a signal is detected only in TK+ cells grown in the presence of BrdU. (C) Relative BrdU levels in known amounts of fully substituted reference DNA (left) and total yeast DNA after one round of DNA replication with 400 µg/ml BrdU (right). Purified reference and test DNAs were denatured by boiling, spotted on a membrane and BrdU quantitated using anti-BrdU and fluorescent secondary antibodies. After correcting for one-strand synthesis, the ratio of BrdU versus dT incorporated in vivo is 0.1. Mean values ± SD are shown for three independent experiments.
Figure 2
Low level BrdU incorporation does not affect cell cycle progression. (A) TK+ yeast cells (E1000) were grown for 2 h at 25°C in the presence of increasing amounts of BrdU (0–1600 µg/ml). After PFGE DNA was stained with SybrGold (left), transferred to nitrocellulose and the level of BrdU incorporated into chromosomes quantitated using anti-BrdU and fluorescent secondary antibodies. (B) Total incorporation is proportional to the amount of BrdU present in the medium (dark gray bars). The incorporation obtained with a concentration of 400 µg/ml BrdU in the medium affects neither the doubling time (light gray bars) nor the growth curves (C), nor cell cycle distribution (D) of the cultures. A slight increase in doubling time is seen at higher BrdU concentrations.
Figure 3
BrdU is incorporated into chromosomal DNA during S phase. (A) Small unbudded TK+ yeast cells (E1000) were isolated by centrifugal elutriation and incubated at 25°C in the presence of either 400 µg/ml BrdU or 20 µCi/ml [3H]uracil. α-Factor was added to the cultures at 150 min to prevent a second round of DNA replication. Samples were collected every 15 min and incorporation of BrdU and 3H into DNA was measured as described in Materials and Methods. 3H c.p.m. values (25 × 103 at 100%) are the means ± SD of three independent cell samples. (B) Cell cycle progression in the presence of BrdU was monitored by FACS analysis and was indistinguishable from cells grown without BrdU (data not shown).
Figure 4
Pulse–chase incorporation of BrdU during S phase. (A) TK+ cells (E1000) arrested in G1 with α-factor for 2 h were released by addition of 50 µg/ml pronase in medium containing 200 µg/ml BrdU at 25°C. The culture was split in two and cells were either collected at 10 min intervals during the following S phase (–chase) or were chased with a 10-fold excess of thymidine (dT) at the same time points (+chase). In the latter case, cells were only harvested in G2 (100 min). (B) FACS profile of the pulse–chase experiment. (C) PFGE analysis of the +chase and –chase experiments. The electrophoresis was performed as described in Materials and Methods and the gel was stained with ethidium bromide. (D) Detection of BrdU incorporation. Southern blotting and detection were performed as described in Figure 1, except that a secondary antibody coupled to HRP was used and revealed with an ECL reaction (Amersham). (E) Quantification of BrdU signals on the Southern blot shown in (D). As only fully replicated chromosomes enter the gel in the –chase experiment, the shift on the _x_-axis between the two curves indicates S phase duration.
Figure 5
Analysis of S phase progression in cell cycle mutants. E1000 (wild-type), E742 (_clb5_Δ_clb6_Δ) and E996 (cdc6-1) cultures were arrested in G1 phase with α-factor and released by addition of pronase in medium containing 400 µg/ml BrdU. cdc6-1 cells were pre-synchronized in G2/M with nocodazole and maintained at 37°C in order to inactivate Cdc6p prior to α-factor release. (A) Genomic DNA was extracted from agarose plugs identical to those used in (B) and the total amount of BrdU incorporated was quantitated as described in Materials and Methods. (B) Percentage of S phase completion estimated from re-entry of BrdU-labeled chromosomes into the PFGE gel. (C) Kinetics of DNA replication as seen by FACS analysis of DNA content in the three cultures.
Figure 6
Immunodetection of BrdU incorporated into intact yeast nuclei. (A) Exponentially growing _GPD_–_TK_7× ORC2_–_myc cells (E1018) were labeled with 400 µg/ml BrdU for 30 min and treated for immunofluorescence as described in Materials and Methods. All cells show a punctate Orc2 pattern (red) while only those in S phase during the pulse display BrdU incorporation sites (green). (B) E1018 cells were released from α-factor arrest into medium containing BrdU and 0.2 M HU. In this case all cells are BrdU-positive. (C–E) Deconvoluted optical sections of an S phase cell labeled as described in (A). Stepped 0.2 µm _z_-axis images were acquired with MetaMorph on a wide field microscope and deconvoluted using the Huygens software. The gallery shows that ORC foci (C) and BrdU foci (D) are found within the nucleus interior, but co-localize only partially (E). Bar 1 µm.
Figure 7
Analysis by DNA combing of replicon size and distribution in HU-arrested cells. (A) TK+ yeast cells (E1000) were arrested in G1 with α-factor and released in medium containing 0.4 mg/ml BrdU and 0.2 M HU for 90 min. Cells were embedded and lysed in agarose plugs to preserve DNA integrity. Plugs were digested with agarase and chromosomal DNA was stained with YOYO-1, combed on silanized coverslips and observed by microscopy as described in Materials and Methods. (B) Detection of BrdU-substituted regions on combed chromosomes using anti-BrdU (DAKO) and Alexa 488 antibodies. The bar (20 kb) was derived from size measurements of DNAs of known length. (C) Size distribution (in kb) of the BrdU signals. (D) Histogram of distances between the centers of adjacent BrdU-labeled regions. (E) Quantification of the overall BrdU incorporation in HU-arrested cells. E1000 cells were released from α-factor arrest in the presence (filled circles) or absence (open circles) of HU. For each time point the relative amount of incorporated BrdU was determined and normalized to genomic DNA as described in Materials and Methods. HU-arrested cells incorporate 23% of the BrdU incorporated during a complete round of DNA replication. (F) Model for the distribution of active origins in HU-arrested cells. Average IOD derived from total BrdU incorporation (model 1), replicon size measurements (model 2) and a model reconciling both types of calculation (model 3) (see text for details).
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