Laboratory of Molecular Gerontology, National Institute on Aging, NIH
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Aggarwal, M., Brosh Jr., R. M. Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System . J. Vis. Exp. (37), e1639, doi:10.3791/1639 (2010).
1. Yeast Strains
Strains with wild-type SGS1 TOP3 (WT; W303-1A, genotype, MATa ade2-1 canl- 100 his3-11,15 leu2-3,112 trpl-l ura3-1) , a sgs1 mutant (W1292-3C; genotype MATa SUP4-o::URA3 sgs1-25 ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 rad5-535) and a sgs1 top3 mutant ( W1058-11C, genotype, MATa SUP4-o::URA3 sgs1-25 top3-2::HIS3 ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 rad5-535) have been characterized  and were kindly provided by Dr. Rodney Rothstein (Columbia University).
2. Plasmid DNA constructions
The WRN gene was cloned in YEp112SpGAL following the scheme as discussed.
Yeast cultures were grown using standard protocol and transformations were performed using a Lithium Acetate-based protocol by Gietz et al . Briefly, the following steps were performed.
4. Genetic analysis of the slow growth phenotype restoration in WRN transformed sgs1 top3 strain.
1. Streak analysis:
2. Liquid culture analysis:
5. Genetic analysis of hydroxyurea or methylmethane-sulfonate sensitivity in WRN transformed yeast strains.
6. Cell cycle distribution of WRN transformed sgs1 top3 strain.
7. Western blot analyses.
1. To determine the protein expression of WRN or the WRN variants in transformed sgs1 or sgs1 top3 strains, lysates were prepared by the following method and proteins analyzed by immunblotting.
2. To determine the level of expression of WRN protein under the influence of varying concentrations of galactose, cultures were processed as follows.
3. Expression of WRN or WRN mutant proteins was determined by Western blot using a WRN mouse monoclonal antibody directed against an epitope in a purified C-terminal fragment of WRN  (1:1000, Spring Valley Labs).
4. Primary antibody incubation was followed by incubation with a secondary (horseradish peroxidase-conjugated) antibody (1:5000). Blots were processed with an ECL Plus Western detection system per the manufacturer's protocol.
5. For quantitative Western blot analysis, increasing concentrations of purified recombinant His-tagged full-length WRN protein were included on gels such that upon quantification of the blot a standard linear curve could be generated. Further, cell extract from equivalent amount of cells or 20 μg of the yeast lysate samples were loaded on gels to estimate the concentration of WRN.
6. ImageQuant analysis was then performed on gels and standard linear curve was generated.
7. Concentration of WRN protein at varying gal concentrations was estimated using standard linear curve generated above.
8. Representative Results
All strains used in this study are Trp auxotrophs. Therefore, sgs1, sgs1 top3 and wild type W303-1A strains transformed with YEp112SpGAL or YEp112SpGAL-WRN were selected on the basis of their ability to grow in presence of SC minus Trp media. To examine the effect of WRN expression on the growth of transformed sgs1 top3 mutant cells, the cultures were streaked onto plates containing SC minus Trp media with 2% gal to induce WRN expression. Top3 decatenates intertwined DNA molecules generated by Sgs1 helicase during replication [1, 2]; therefore, in the absence of Top3, torsional stress is not relieved resulting in slow growth and hyper-recombination. The genetic function of sgs1 is to suppress the slow growth phenotype of a top3 mutant. If WRN could substitute for SGS1 in genetic interaction with Top3, restoration of top3 slow growth phenotype in sgs1 top3 would be expected.
As shown in Figure 2A, the WRN transformed sgs1 top3 strain grew significantly more slowly compared to the vector transformed sgs1 top3 strain. sgs1 top3 transformed with the YEp112SpGAL-SGS1 plasmid was included as a positive control (Figure 2A), demonstrating that wild-type Sgs1 expressed in the sgs1 top3 mutant was able to genetically complement the growth phenotype. The transformed sgs1 top3 strains grew similarly in the absence of gal as shown for 2 days (Figure 2A). Expression of WRN had no effect on the growth of parental wild-type (W3031A) or sgs1 strains (Figure 2B), indicating that the effect of WRN on cell growth was specific to that observed in the sgs1 top3 mutant background. Genetic studies performed using WRN variants demonstrated that WRN helicase but not exonuclease activity was required for the restoration of top3 growth phenotype (Figure 3B). A naturally occurring missense polymorphism in WRN that interferes with helicase activity abolished its ability to restore top3 slow growth phenotype.
top3 mutant strains are delayed in the late S/G2 phase of the cell cycle , a characteristic that may account for their slow growth. Mutation of the SGS1 gene in the top3 background suppresses the delay in the S/G2 phase of the cell cycle. If WRN expression could restore the delay in the S/G2 phase of the cell cycle in sgs1 top3, an elevated population of large budded cells with undivided nuclei would be expected for sgs1 top3 mutant cells expressing WRN. As shown in Figure 4, the percentage of large budded cells was higher for sgs1 top3/WRN than sgs1 top3/vector, suggesting the restoration of the S/G2 delay characteristic of top3.
The sgs1 top3 double mutant is less sensitive to MMS or HU than the top3 single mutant . Since WRN affected cell growth of sgs1 top3, we next examined its effect on sensitivity to methylmethane sulfonate (MMS, an alkylating agent) and hydroxyurea (HU, a replication inhibitor). sgs1 top3 / WRN displayed sensitivity to both the drugs comparable to sgs1 top3/ SGS1 (Figure 5). Genetic analysis of WRN variants revealed that WRN helicase /ATPase, but not WRN exonuclease activity, was required for its effect on sensitivity to MMS and HU.
Figure 1. Schematic presentation for cloning WRN / WRN variants in YEp112SpGAL vector. Please click here for a larger version of figure 1.
Figure 2. WRN expression in sgs1 top3 restores the slow growth phenotype of top3. Panel A, sgs1 top3 strain transformed with YEp112SpGAL or YEp112SpGAL WRN were streaked on an SC-Trp plate containing either 2% glu or 2% gal. As a control sgs1 top3 strain transformed with YEp112SpGAL SGS1 was streaked on both the plates. Plates were incubated at 30°C for 2 days and then photographed. Panel B, Wild type parental strain W303-1A or sgs1 strain transformed with YEp112SpGAL or YEp112SpGAL WRN were streaked on an SC-Trp plate either containing 2% glu or 2% gal. Plates were incubated at 30°C for 4 days and then photographed. Composition of the plates was as in Panel A and Panel B respectively. Please click here to see a larger version of figure 2.
Figure 3. WRN ATPase/helicase, but not exonuclease activity, is required to restore the slow growth phenotype of top3 in sgs1 top3 background. sgs1 top3 strain transformed with ATPase/helicase-dead (YEp195SpGAL WRN K577M), exonuclease-dead (YEp195SpGAL-WRN E84A), RQC mutant (YEp195SpGAL-WRN K1016A), or polymorphic mutant (YEp195SpGAL-WRN R834C) was streaked on SC-Trp plates containing either 2% glu (Panel C) or 2% gal (Panel B). Plates were incubated at 30°C for 2 days and then photographed. Composition of the plates was as in Panel A. Please click here to see a larger version of figure 3.
Figure 4. WRN expression induces S/G2 arrest in sgs1 top3 cells. Logarithmically growing cultures of sgs1 top3 strain transformed with YEp112SpGAL, YEp112SpGAL WRN, or YEp112SpGAL SGS1, and the vector-transformed wild-type parental strain were induced at 2% gal concentration for 6 h. Cultures were harvested, processed for DAPI staining as described in Materials and Methods and were observed using Axiovert 200 M microscope (Zeiss; 100x lens). Shown is the DAPI staining of the sgs1 top3 transformed with YEp112SpGAL (upper left) and with YEp112SpGAL WRN (upper right). Arrows show cells with undivided nuclei. Distribution of the cells in G1 (single cells) and S/G2 (budded cells) is shown in lower panel. Please click here to see a larger version of figure 4.
Figure 5. Effect of WRN expression on the MMS and HU sensitivity of sgs1 top3 strain. Logarithmically growing cultures of sgs1 top3 strain transformed with YEp112SpGAL , YEp112SpGAL WRN, exonuclease-dead (YEp112SpGAL-WRN E84A), ATPase/helicase-dead (YEp112SpGAL WRN K577M), RQC mutant (YEp112SpGAL-WRN K1016A), polymorphic mutant (YEp112SpGAL-WRN R834C), YEp112SpGAL SGS1 and vector transformed wild type parental strains were spotted in a ten-fold serial dilutions onto SC-Trp plates containing glu or gal and either MMS or HU at the indicated concentrations. Plates were incubated at 30°C for 2 days (control plates) and 4 days (MMS and HU plates) and then photographed. Please click here to see a larger version of figure 5.
One of the strengths of using yeast as a model system is the availability of mutants in defined DNA replication and repair pathways that are conserved between yeast and humans. Further selection of transformants harboring the specific genes is easy and reliable as the laboratory strains are auxotrophic mutants and vectors with auxotrophic markers are readily available. Using these vectors the expression of gene products can be regulated by placing them under the control of an inducible promoter (e.g., gal inducible promoter, etc.). Considering these advantages, we developed a yeast based model system to study the functional requirements of the human WRN gene defective in Werner Syndrome in a pathway that is potentially conserved between human and yeast. Using the described approaches, we provided the first evidence that WRN can function in a genetic pathway that affects top3-related phenotypes. Our observations prompt further investigation of the possibility that WRN functionally interacts with Top3α in human cells during cellular DNA replication or recombination. Conceivably, in a BLM-impaired condition, WRN may partially substitute for BLM through its protein partnership with a topoisomerase.
This work was supported in full by the Intramural Research program of the NIH, National Institute on Aging. We thank Dr. Rodney Rothstein (Columbia University) for the yeast strains and Dr. Brad Johnson (University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania) for the SGS1 expression plasmid.