Table Of Content

MMeetthhooddss iinn MMoolleeccuullaarr BBiioollooggyy TTMM VOLUME 113 DDNNAA RReeppaaiirr PPrroottooccoollss EEuukkaarryyoottiicc SSyysstteemmss EEddiitteedd bbyy DDaarryyll SS.. HHeennddeerrssoonn HHUUMMAANNAA PPRREESSSS Technical Notes UV-A, UV-B, and UV-C: This terminology, which divides the ultraviolet (UV) spectrum into three wave bands, was first proposed in 1932 by the Ameri- can spectroscopist William Coblentz and his colleagues to begin to address the problem of standardizing the measurement of UV radiation used in medi- cine(1,2). Each spectral band was defined “provisionally” and “approximately” by the absorption characteristics of specific glass filters as follows: UV-A, 400–315 nm; UV-B, 315–280 nm; UV-C, <280 nm (1). Although based on physical specifications, these definitions were influenced by knowledge of other UV phenomenology, including biological effects and physical proper- ties. For example, wavelengths in the UV-B band were known to have potent erythemic effects, and wavelengths below 290 nm were known to be absent from sunlight (2) (because they are absorbed by stratospheric ozone). More- over, the germicidal effects of UV-C wavelengths (principally around 266 nm) from artificial sources were well-recognized (3). Today, the spectral bands implied by these terms may be found to vary from Coblentz’s original definitions, depending on the discipline. Environmental photobiologists, for example, generally use the following definitions: UV-A, 400–320, UV-B, 320–290, and UV-C, 290–200 (4). Relative centrifugal forces: The g-forces listed in this book are calcu- lated for the maximum radius unless stated otherwise. For microcentrifuges similar to Eppendorf’s 5410 and 5415 C models, maximum rotational speed (14,000 rpm) corresponds to ~12,000g and ~16,000g, respectively. References 1. Coblentz, W. W. (1932) The Copenhagen meeting of the Second International Congress on Light. Science76, 412–415. 2. Coblentz, W. W. (1930) Instruments for measuring ultraviolet radiation and the unit of dosage in ultraviolet therapy. Br. J. Radiol.3, 354–363. 3. Gates, F. L. (1930) A study of the bactericidal action of ultra violet light. III. The absorption of ultra violet light by bacteria. J. Gen Physiol.14, 31–42. 4. Diffey, B. L. (1991) Solar ultraviolet radiation effects on biological systems. Phys. Med. Biol.36,299–328. xix Checkpoint Mutant Screen in S. pombe 1 1 Isolation of DNA Structure-Dependent Checkpoint Mutants in S. pombe Rui G. Martinho and Antony M. Carr 1. Introduction Eukaryotic cells have the ability to influence progression through the cell cycle in response to internal and external inputs of “information”. They do so by using feedback control mechanisms able to arrest mitosis in response to different cellular events. Such active mechanisms capable of influencing the timing of cell-cycle events have been called “checkpoints” (1,2). Cells arrest progression through the cell cycle if they fail to complete DNA replication or if their DNA is damaged. The S-phase/mitosis (S-M) checkpoint plays a key role in the maintenance of the interdependency between S-phase and mitosis. Wild- type Schizosaccharomyces pombe (fission yeast) cells arrest cell-cycle progression in response to a DNA replication block, such as that induced by hydroxyurea (HU), but continue to grow in size, since they are still metaboli- cally active. These cells are observed to have an elongated phenotype. Mutants have been isolated in S. pombe that have lost the S-M checkpoint and do not prevent mitosis if DNA replication during the previous S-phase is incomplete (3–7). S-M checkpoint mutants do not delay cell-cycle events after exposure to HU, and will enter mitosis with unreplicated DNA. As a consequence, the elongated phenotype seen for wild-type cells is absent in checkpoint mutants. In- stead, these mutants show a characteristic “cut” phenotype, where a cell has entered an abortive mitotic event followed by the formation of a septumthrough the nucleus. In these small dead cells, the nucleus is frequently cut in two by the septum and/or spread unevenly between both daughter cells. S-M checkpoint mutants show very low viability in the presence of HU or any other cir- cumstance that may delay S-phase progression (e.g., in combination with a thermosensitive DNA replication mutant). From:Methods in Molecular Biology, Vol. 113: DNA Repair Protocols: Eukaryotic Systems Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ 1 2 Martinho and Carr Several of these S-M checkpoint mutants are also unable to arrest the cell cycle in response to DNA damage (6,7). The DNA damage checkpoint arrests the cell cycle in a dose-dependent manner after exposure to DNA-damaging agents. It is believed that these delays provide additional time for cells to repair the damaged genetic material before key transitions are attempted (between G1 and S-phase, G2 and mitosis, or during DNA replication). Several different mutants have been isolated in S. pombethat are defective in the DNA damage checkpoint. In contrast to wild-type cells, which arrest the cell cycle immedi- ately after DNA damage (and display the previously described elongated phenotype), most of these checkpoint mutants do not show any delay in cell-cycle events after exposure to DNA-damaging agents. One or two of these mutants are only partially defective (e.g., rad24). As seen for the S-M checkpoint mutants, the DNA damage checkpoint mutants can also show a cut phenotype where cells enter unrestrained mitosis with damaged DNA. All these mutants are highly sensitive to DNA-damaging agents. We refer to these two checkpoints (S-M and DNA damage) as DNA structure-dependent checkpoints. The existence of many genes whose function is required for both checkpoint controls suggests a significant overlap between these two pathways. The struc- tural identity between the checkpoint proteins from fission and budding yeast suggests that these pathways have analogs in mammalian cells. This is sup- ported by the growing number of human genes found to be homologous to yeast checkpoint genes (8). We describe below methods for: 1. Generating mutants of S. pombe. 2. Screening those mutants for putative DNA structure-dependent checkpoint defects. 3. Distinguishing between S-M and DNA damage checkpoint deficiencies. 4. Further characterizing checkpoint mutants. 2. Materials 2.1. Media 1. Yeast extract medium (YE): 5 g/L Difco (Detroit, MI) yeast extract, 30 g/L glu- cose, supplemented as required with 100 mg/L leucine, adenine, lysine, uracil, and histidine. 2. Yeast extract agar medium (YEA): YE plus 20 g/L Difco agar. 3. YEP: YE plus 20 g/L Difco Bacto-peptone. 4. Phloxin B agar (YEA + P): YEA plus 0.02 mg/mL Phloxin B (Sigma, Dorset, UK). Phloxin B is stored as a stock solution at 20 mg/mL. It should be added after the medium is autoclaved and cooled. 5. YEA + P with HU: YEA + P containing 10 mMHU. HU is kept as a 1 Mstock solution stored at –20°C. It is filter-sterilized and added to autoclaved, cooled medium. Checkpoint Mutant Screen in S. pombe 3 2.2. Additional Reagents and Equipment 1. Ethyl methanesulfonate (EMS is listed in Sigma’s catalog as methanesulfonic acid ethyl ester). 2. 254-nm UV source (e.g., germicidal lamp). 3. 4,6-Diamino-2-phenylindole (DAPI) (Sigma). 4. Calcofluor (Sigma).(Also known as “Flourescent Brightener 28.”) 5. Replica plating block. 6. Whatman filters, no. 1, 150 mm. (It is not necessary to sterilize filters from a new box.) 7. Hemocytometer. 8. Light microscope equipped with a 20× long-distance working objective. 9. Fluorescence microscope. 3. Methods 3.1. EMS Mutagenesis Optimization of the mutagenesis procedure is an empirical process. Prelimi- nary mutagenesis studies should be performed in order to find the experimental conditions that give the highest number of potentially interesting mutants with a reasonable level of survival (not <10%). For example, a good mutagenesis procedure using wild-type cells should give approximately one DNA damage checkpoint mutant/1000 surviving cells, with a survival rate of about 10–20%. EMS has been previously used with much success for checkpoint mutant screens in fission yeast, although alternative mutagenesis procedures (e.g., using UV irradiation, see Note 1) should be considered, because gene targets may differ. This may be an important consideration if the desired mutants are rare or difficult to isolate. 1. Prepare a fresh 50-mL culture of log-phase cells (OD = 0.2–0.4) growing 600 in YEP. 2. Collect the cells by centrifugation (~2000g) for 2 min and resuspend in 1 mL of YEP medium containing EMS (2.5–3% v/v). Ensure the EMS is completely dissolved. 3. Incubate with shaking at room temperature for 2 h. 4. Wash the cells several times with fresh medium and plate enough cells on YEA plates to give approx 500 colonies/plate. This should be around 5000 cells/plate, assuming a survival rate of approx 10%. 5. Incubate the plates at 27°C. (Different permissive conditions may be required for the isolation of thermosensitive mutants.) 3.2. Identification of S-M Checkpoint Mutants (see Note 2) The HU sensitivity screen has been one of the most efficient and suc- cessful experimental approaches for identifying new DNA structure checkpoint mutants, since it provides easily definable phenotypes. HU is a 4 Martinho and Carr powerful inhibitor of the ribonucleotide reductase enzyme that catalyzes the rate-limiting step in the production of deoxyribonucleotides needed for DNA replication. 1. Replica plate the mutagenized colonies from Subheading 3.1. onto two plates, one YEA and one YEA + P with HU as follows: Press the master plate against the replica-plating block covered with a Whatman filter. Gently remove the plate in such way that a replica of colonies from the master plate remains on the filter. Transfer the cells to replica plates by repeating the procedure. Remove excess cells from the replica plates by pressing each plate against a clean filter. Incubate at 27°C for 24–48 h. 2. Dead colonies on HU-containing YEA + P plates will appear red in color. Phloxin B stains dead cells red, but is actively excluded from live cells. Most of these dead colonies when observed under the microscope will contain many highly elongated dead cells (stained red). The HU-sensitive S-M checkpoint mutants will have a different morphology characterized by small dead cells and no elongation. 3. From the YEA master plate, pick cells that correspond to phenotypically interesting dead colonies and patch onto a new YEA plate. 4. Confirm the phenotype of these patches by replica plating again onto YEA and YEA + P with HU. Incubate the plates at 27°C for 48 h. Discard those mutants that do not show a reproducible phenotype (seeNotes 3 and 4). 3.3. Identification of DNA Damage Checkpoint Mutants (see Note 5) The isolation of DNA damage checkpoint mutants is more difficult than the identification of S-M checkpoint mutants, because the phenotypes observed during the screen are not as accurate as with cells treated with HU (particularly if UV radiation is used as the selective agent). Since most S-M checkpoint mutants are also deficient in the DNA damage checkpoint, the following experimental procedure should also be used to check any new S-M checkpoint mutant previously isolated. This screen should be performed simultaneously with the HU screen by including an extra replica plating. 1. Replica-plate the mutagenized colonies onto two plates, one YEA and one YEA+ P, as described in step 1 of Subheading 3.2.Make sure the excess of cells is removed from both plates. 2. UV-irradiate the YEA-P plates with 200 J/m2. 3. Incubate at 27°C for 48 h. 4. Dead colonies on the UV-irradiated plates will appear as red “spots”. Most of these dead colonies when observed under the microscope will contain lots of dead cells (stained red), most of which will show some degree of elongation (see step 2, Subheading 3.2.). In the DNA damage checkpoint mutants, this elongated phenotype will be absent or greatly reduced. Checkpoint Mutant Screen in S. pombe 5 5. Pick from the YEA master plate those cells that correspond to phenotypically interesting dead colonies, and patch onto a fresh YEA plate. 6. Confirm the phenotype of these patches by replica plating again onto YEA and YEA + P media. Irradiate the YEA + P plates, and incubate at 27°C for 48h.Discard any mutants that do not show a reproducible phenotype (seeNotes 3 and 4). 3.4. Survival Analysis In order to have a clear picture of the nature of the mutants isolated in the screen, it is useful to determine their survival response to DNA-damaging agents and HU, and to do a microscopic analysis of cell morphology. This information will help to classify the mutants into groups, and identifies interesting and desired phenotypes. 3.4.1. HU Survival Curves (seeNote 6) 1. Determine the cell number of a fresh exponentially growing culture using a hemocytometer. 2. Dilute to a cell density of ~5000 cells/mL in YEP. 3. Add HU to the diluted cell culture to a final concentration of 10 mM. 4. Incubate the culture at 30°C, take a 100-µL sample at different time-points (0, 1, 2, 3, 5, 7, 10 h) and plate onto YEA plates. 5. Incubate at 27°C for 72 h. 6. Count the colonies, and calculate the percent survival by comparing with the time-zero control plate. 3.4.2. UV Survival Curves (seeNotes 7 and 8) 1. Follow steps 1 and 2 of Subheading 3.4.1. 2. Plate 100-µL aliquots of the diluted cell culture onto each of 16 YEA plates (500 cells/plate). 3. UV-irradiate the plates using the following doses: 0, 25, 50, 100, 150, 200, 250, and 300 J/m2. All UV treatments should be done in duplicate. 4. Incubate the plates at 27°C for 72 h. 5. Count the number of colonies, and calculate the percent survival by comparing with the nonirradiated control plates. 3.5. Microscopic Analysis The morphology of the mutant cells and their nuclei after exposure to HU can be studied using the DNA-specific fluorescent dye DAPI and an additional dye, calcofluor, that stains material of the septum. The cells are then examined by fluorescence microscopy to determine their morphology. 1. Take cells from an exponentially growing culture, and incubate in YEP containing 20 mM HU at 30°C. 6 Martinho and Carr 2. Take 100-µL samples of cells at 2, 6, and 18/24 h. 3. Collect the cells by centrifugation, wash once in water, resuspend in 10 µL water, and fix in 200 µL of methanol. 4. Spot 10 µL of the fixed sample onto a glass slide, and air-dry for 5 min. 5. Pipet onto a cover slip 5 µL of a water, containing DAPI stain (0.1 µg/mL) and calcofluor (0.5 µg/mL), and gently press against the dried fixed cells on the glass slide. 6. Examine the cells using a fluorescent microscope, and determine the percentage of each phenotype (cut and elongated) for each sample (seeNote 9). 4. Notes 1. Alternative mutagenesis protocol using UV radiation: a. Prepare a fresh culture of log phase cells (as described in step 1,Subheading3.1) b. Plate enough cells onto YEA plates to give ~500 cells per plate surviving mutagenesis (1000-2000 cells per plate assuming a survival rate close to 25–50%). c. Make sure the surface of the plate is well dried, remove the lid and UV irradiate. The UV dose for wild-type cells is ~300 J/m2. d. Incubate the cells as described in step 5 of Subheading3.1. 2. Since the most obvious screens are already very close to saturation, any attempt to isolate new genes involved in the DNA structure checkpoint response should be designed with great care, and specific objectives and different targets decided in order to avoid isolating previously cloned genes. For example, a cdc17(DNA ligase) mutant can be used in a screen comprising synthetic lethality following a transient shift to the restrictive temperature, or a 48-h incubation at the semipermissive temperature. The DNA ligase thermosensitive mutant when incubated at the restrictive temperature (35.5°C) is defective in the ligation of Okazaki fragments during replication. At the restrictive temperature, the cdc17 mutant arrests in S-phase, elongates, and slowly loses viability. This late S-phase arrest is distinct from early S-phase arrest caused by HU. Mutations abolishing the S-M checkpoint in a cdc17background will make the double mutants highly sensitive to elevated temperatures. Double mutants will rapidly become nonviable after a brief incubation at the restrictive temperature (“transient temperature sensitivity”) or a long incubation at the semipermissive temperature, since they will enter an abortive mitotic event with unreplicated DNA, displaying a cut phenotype. In some aspects, screens using the cdc17 genetic background mimic the HU mutant screen, but subtle differences exist that may be useful for the isolation of new checkpoint mutants. a. Replica plate the mutagenized cdc17colonies onto two plates (one YEA and one YEA + P) as described in Subheading 3.2. b. Incubate the YEA master plate at 27°C for 48 h, and the YEA + P plate first at 35.5°C for 9 h and then at 27°C for 48 h, or incubate the YEA master plate at 27°C for 48 h and the YEA + P plate at 31.5°C (semipermissive temperature) for 48 h. Checkpoint Mutant Screen in S. pombe 7 c. Identify those dead colonies on the YEA + P plate comprised of cells with a cut phenotype. Isolate the corresponding cells from the YEA master plate, and patch onto a new YEA plate. d. Confirm the phenotype of these patches by replica plating again onto YEA and YEA + P, repeating step b. e. Discard those mutants that do not show a reproducible phenotype. The 9-h incubation of the cdc17 mutant at 35.5°C (or 48 h at the semipermissive temperature of 31.5°C) reduces the viability of the single mutant, but colonies still form. A double mutant composed of cdc17and any S-M checkpoint mutant will be nonviable and incapable of forming colonies under these conditions. The use of DNA replication mutants like cdc20 (DNA polymerase (cid:161)) that arrest in early S-phase (cdc17 arrests in late S-phase), is also a potentially useful approach since it may uncover different aspects of the S-M checkpoint pathway. 3. Genetic analysis of checkpoint mutants: To ensure that the phenotype seen in each mutant is the outcome of a single gene mutation and not the result of the interaction between two different genetic mutations, it is essential to backcross each mutant three times with wild-type cells. If after this process the phenotype is retained, it is reasonable to assume that only one gene is responsible for it. In addition these backcrosses have the important effect of ensuring a clean genetic background. 4. Most mutant screens target particular genes preferentially in such a way that many of the generated mutants may be identical (e.g., rad3 mutants constitute up to 50% of the S-M and DNA damage checkpoint mutants isolated to date). To avoid unnecessary duplication of work by characterization of two identical checkpoint mutants, it is recommended that mutants be crossed to one another and to known checkpoint mutants with similar phenotypes. If the two strains used in a given cross are allelic, then wild-type cells will not be generated from this cross. Note, that if two different genes are closely linked, wild-type cells may be absent or rare. However, linkage between two different nonallelic mutants with a similar phenotype is very rare. 5. Alternative procedure: The use of (cid:97)-rays in the isolation of DNA damage checkpoint mutants will primarily isolate mutants deficient in G2-M arrest, since this transition is the most critical in cells exposed to ionizing radiation. The experimental procedure is essentially the same as the one described in Subheading 3.3. for the isolation of UV-sensitive checkpoint mutants. A (cid:97)-ray dose of approximately 1000–1500 Gy is required. 6. An alternative HU survival test: the spot test. a. Determine the cell density of an exponentially growing culture using a hemocytometer. b. Dilute each culture to four different concentrations (107, 106, 105, and 104 cells/mL) in rich medium. c. Make three YEA + P plates containing the following concentrations of HU: 3, 5, and 7.5 mM. 8 Martinho and Carr d. Spot 2 µL of each diluted strain onto YEA + P plates containing the different concentrations of HU so that an increased dilution of the same strain is spotted across the plate. Different cell strains should be spotted in parallel lines on the same plate, so that comparisons of their HU sensitivity can be made. e. Incubate the plates at 27°C for 72 h. f. Compare the levels of growth. The spot test and the HU survival analysis described in Subheading 3.4.1. may give different results. This is because the spot tests measures an adaptive response to low concentrations of HU, whereas the survival curves measure a survival response to acute exposure to high concentrations of HU. 7. Alternative procedure: (cid:97)-ray survival curves. The experimental procedure is similar to the one described in Subheading 3.4.2. for testing survival to UV radiation. The plates should be irradiated at the following doses: 0, 50, 100, 200, 400, 500, 1000, and 1500 Gy. If the (cid:97)-ray source has a small irradiation chamber the cells should be diluted to the correct cell density (5000 cells/mL), irradiated and only then plated (as described in Subheading 3.4.2.). 8. Alternative procedure: EMS survival curves. The experimental procedure is similar to the one described in Subheading 3.4.1. for HU survival curves. Incubate the cells in medium containing 2% (v/v) EMS, and take samples as described for determining HU survival. 9. Most DNA damage checkpoint mutants become sensitive to HU at high concentrations or after long incubations, but under standard treatment conditions, they have a normal checkpoint response and are not particularly sensitive to HU. Non- checkpoint DNA repair mutants, when incubated with DNA-damaging agents die with a highly elongated phenotype, because they are unable to repair the DNA damage. Some extremely sensitive DNA repair mutants die with no elongation at normal doses of mutagens. This is because they cannot undertake transcription. At very low concentrations of DNA-damaging agents, such mutants will display a highly elongated phenotype. Acknowledgment We wish to thank Nicola Bentley for helpful comments. References 1. Murray, A. W. (1992) Creative blocks: cell cycle checkpoints and feedback controls.Nature359, 599–604. 2. Hartwell, L. and Weinert, T. (1989). Checkpoints: controls that ensure the order of cell cycle events. Science246, 629–634. 3. Enoch, T., Carr, A. M., and Nurse, P. (1992) Fission yeast genes involved in cou- pling mitosis to completion of DNA replication. Genes Dev. 6, 2035–2046. 4. Saka, Y. and Yanagida, M. (1993) Fission yeast cut5, required for S-phase onset and M-phase restraint, is identical to the radiation-damage repair gene rad4+.Cell 74, 383–393.