The DNA mismatch repair (MMR) pathway corrects specific types of replication errors caused by DNA polymerase slippage and is critical for maintaining genomic integrity. Given its importance, the canonical genes of the MMR pathway are highly conserved among different species including Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. Microsatellite (MS) sequences are composed of homopolymers and tracts of di- or trinucleotide repeats among others. Defective MMR function increases the rate of insertion and deletion (indels) mutations in microsatellites and this molecular phenotype is commonly referred as microsatellite instability (MSI). In H. sapiens, the consequences of defective DNA MMR is dramatically apparent in the Mendelian cancer syndrome, hereditary nonpolyposis colorectal cancer (HNPCC), otherwise known as Lynch Syndrome [
In S. cerevisiae, the MMR genes encode for the proteins Msh2p, Mlh1p, Msh3p, Pms1p and Msh6p, all of which make up two basic protein complexes that mediate MMR. Also, there is substantial experimental evidence that EXO1, a 5′→3′ exonuclease, is involved in MMR given evidence of physical interactions with Msh2p and Mlh1p [
As previously described, we constructed a series of plasmids (the pHJ series) in which a segment of a tumor suppressor cDNA sequence with a coding microsatellite is placed upstream and in-frame of the selectable marker gene URA3 [
Functional genomics screen
For the MSI functional genomics screen, we used the plasmids pHJ-9 (MS-positive experimental vector) and pCI-HA (MS-negative control vector). The pHJ9 plasmid is derived from pCI-HA which is a low copy number centromeric vector, has a unique BamHI site located upstream of URA3 and contains an additional LEU2 marker for plasmid retention [
We used the diploid yeast pool representing homozygous deletion mutant strains for 4,728 non-essential genes [
We performed five independent screens with the pHJ-9 and pCI-HA plasmids. The screen has several steps as shown in Figure 2. For each replicate experiment, we transformed either pHJ-9 or pCI-HA into the combined pool of all of the homozygous deletion mutant pool and subsequently plated the cells on synthetic dropout (SD)
plates lacking leucine (Leu-). During the initial growth period on Leu- media, the plasmids undergo replication without selective pressure to maintain the wildtype URA3 and have the opportunity to accumulate mutations in the individual plasmid. After the initial transformation of the deletion mutant pool, we observed over 7,000 colonies per a replicate experiment. After three days of growth on SD-leu plates, transformed colonies are distinctly visible and we used replica plating to transfer the colonies to a dual selection media plates (Leu-, FOA). After selection on FOA media, we harvested all of the FOAR colonies, extracted genomic DNA from the collected cells, amplified the deletion mutation barcodes with PCR, hybridized the PCR amplicons to the TAG3 barcode microarrays and scanned the arrays post-hybridization [
Analysis of TAG3 barcode microarray data
A normalization procedure on the microarray barcode intensities was applied for all further data analysis [8, 9]. From the array barcode data, we identified the FOAR deletion mutants that showed significant fold change increase compared to pCI-HA control. To assess the false discovery rate (FDR) when analyzing for fold change (FC) differences on the five replicate pairs of experiments the program PaGE 5.1 was used [
by PaGE 5.1, the results in Table 1 include the confidence level that is one minus the FDR. Table 1 lists the most significant results and the average mean intensity for the tag barcodes of the experimental versus the control condition.
The FDR analysis did not yield the msh2 and mlh1 deletion mutants among the top ranked tag barcodes. Among the replicate experiments and array data, we examined at the average fold change for the barcode intensities for the msh2 and mlh1 mutants. No statistically significant FC increase was noted for msh2 but mlh1 did show a FC increase of 2.7 (p
Fluctuation analysis to validate MSI-related mutants and determine mutation rates
Individual validation experiments on the four candidate deletion mutants were carried out using strains that were independently isolated during the original creation of the mutant. Each deletion strain was obtained from separate archived glycerol stocks and the cells were colony purified. These replicate strains had not been part of the original homozygous diploid pool that was used.
Prior to transformation, pHJ-9 was sequenced and confirmed that no MSI mutations existed in the homopolymer tract. pHJ-9 or pCI-HA deletion mutant strains, were individually transformed and selected for transformants on dual selection SD media plates for Leu- and Ura- conditions. The transformed strains were colony purified prior to determining MSI mutation rates. The original diploid wildtype background strain BY4743 and an mlh1 deletion mutant strain were used as a negative control and a positive control respectively.
The mutation rates for each individual strain were measured using the method-of-the-median for fluctuation analysis. This required both pHJ9 (MS-positive experimental) and pCI-HA (control) [
The fold increase of the mutation rate for a given deletion mutant was calculated by dividing the mutation
rate for pHJ-9 in the mutant versus the wildtype background. The mlh1 mutant had a 37.8 fold elevation of mutation rate compared to the wildtype strain. The PAU24 deletion mutant had a 32.1 fold elevation compared to the wildtype strain. The other deletion mutants demonstrated a two-fold or less increase of the mutation rates.
To verify that the PAU24 mutant strain used for these MSI mutation rate experiments had the appropriate deletion of the PAU24 gene, we sequenced the junctions of the deletion cassettes with the adjacent genomic sequence from the PAU24 homozygous deletion mutant. The sequences from the diploid homozygous mutant as well as the used haploid parental strains were amplified. Specific PCR primers were used to amplify out the deletion cassette and the adjacent yeast genomic sequence. These amplicons were subsequently Sanger sequenced. It was confirmed that the specific PAU24 deletion had occurred based on comparing the known genomic sequence flanking the PAU24 ORF and the correctly assigned deletion tag barcode.
DNA sequencing confirmation of de novo MSI mutations in the PAU24 mutant
In the PAU24 background, we determined if FOAR strains had MSI-related indel mutations in the homopolymer (A)10 tract of the pHJ-9 plasmid (MS-positive). First, the plasmids from independent experiments and different FOAR colonies were recovered. Based on these criteria, these plasmids had completely independent mutation events. From these recovered plasmids, the target MS homopolymer tract were sequenced and the mutations in the target MS region were identified. The same recovery procedure and DNA sequencing analysis was also carried out for the mlh1 FOAR strains. The results of the DNA sequencing analysis are summarized in Table 2. 81% of the 58 plasmids, recovered from the mlh1 deletion mutants, had indel mutations in the homopolymer (A)10 tract of pHJ9. 60% of the 40 plasmids recovered from the PAU24 mutant, had indel mutations. Overall, the predominant mutations were 2 bp deletions followed by 1 bp deletions or 1 bp insertions. From transformation of the wildtype strain grown on Leu- media, we also recovered and sequenced ten pHJ-9 plasmids, none of which demonstrated MSI mutations in the homopolymer tract. This eliminated the possibility that any of the pHJ-9 clones had spontaneously developed a MSI mutation. We also sequenced multiple colony- purified clones of pHJ-9 from our original plasmid preparation and did not identify any mutations.
As noted previously, Lynch syndrome or HNPCC is caused by germline mutations in one of several DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6 and PMS2 [
these genes have not been demonstrated to contribute significantly to HNPCC [
To address the question about the contribution of other genes to MSI we used S. cerevisiae as a model organism system. Utilizing the homozygous diploid deletion mutant pool resource that has frequently been used for functional genomics studies in yeast, a screen identifying homozygous deletion mutants that demonstrate a MSI phenotype was developed (4). An experimental plasmid with a homopolymer (A)10 tract that is found in the human TGFBR2 gene and frequently mutated in human MSI-positive colorectal cancers was used. This plasmid models the sequence context that is frequently seen in MSI-positive cancers. We identified a deletion mutant strain of the PAU24 gene that has a MSI phenotype. This finding was confirmed in separate validation experiments using independently derived deletion mutants; the PAU24 deletion mutant shows elevated MSI mutation rate compared to wildtype strains and other deletion mutant strains. Through sequencing rescued plasmids, we also demonstrated microsatellite specific indel mutations in the introduced homopolymer tract from strains showing the appropriate MSI phenotype. In addition, it was confirmed that the PAU24 gene had been appropriately deleted in our experimental mutants.
Relatively little is known about the function of PAU24 in S. cerevisiae. Given its sequence homology, it has been generally classified as a cell wall mannoprotein and a member of the seripauperin multigene family [
One aspect of this genomics screen which may have affected the selection of mutants for MSI is the characteristics of the microsatellite [
There is a previous report of a bystander mutation in the mismatch repair gene MSH3 that caused MSI among a small subset of yeast deletion haploid mutants from the original consortium [
In addition, the bystander MSH3 mutation arose from a single laboratory and a single wildtype haploid strain specific to that laboratory (Angela Chu, Personal Communication 2012). Most notably, these mutants were created by transformation into a haploid (1N) strain, then mating two haploids from the initial transformation to make the homozygous diploid. This approach is substantially more prone to producing bystander mutations with a specific phenotype given the initial use of a haploid (1N) strain. Generally, most of the deletion consortium members started with a diploid strain (2N) transformation followed by dissection for haploids and then mating to creating the diploid mutants. This is the exact reverse of what was done in the case of the bystander MSH3 mutant. The PAU24 mutant strain was not generated by the group that produced the MSH3 background mutant. Furthermore, the YBR301W strain with the PAU24 deletion were made by transformation into the diploid, and dissected to get haploids, which were subsequently mated to make the homozygous diploid. For the MSI analysis, we used deletion mutant replicates derived from separate transformations and matings, thus lessening the chances of a random bystander mutation in a mismatch repair gene. We are continuing to investigate the relations between the PAU24 deletion strain and the MSI phenotype.
Materials and Methods
General genetic methods, plasmids and strains
SD media were obtained from Bio101 Systems (Santa Ana, CA). 5-Fluoroorotic acid (FOA) was obtained from Zymo Research (Orange, CA). The SD-leu-ura plates contained FOA at a concentration of 1 gm per liter and uracil at 50 mg per liter. This concentration was empirically determined to be optimal for selecting FOA resistant (FOAR) colonies. Homozygous diploid deletion strains including PAU24 (strain 37177), skn1 (strain 23773), dnf2 (34182), gyp8 (35646) and the wildtype strain BY4743 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 MET15/met15Δ0 URA3Δ0/URA3Δ0) were obtained from the Stanford Genome Technology Center. The diploid BY4743 was the original strain used in the construction of all deletion mutants [
Functional genomics MSI screen and TAG3 microarray analysis
For each replicate experiment, we transformed either pHJ-9 (MSI-experimental) or pCI-HA (control) into the combined pool of the homozygous deletion mutant pool and subsequently spread the cells on SD-leu plates at 30 °C. The characteristics of the pool are as previously described [
For the array preprocessing two Perl scripts were used as previously described [
Mutation rate analysis
Each deletion strain was obtained from separately archived glycerol stocks and colony purified the cells, making sure to use a replicate deletion mutant strain that had not been part of the original homozygous diploid pool. Using either pHJ-9 or pCI-HA, we individually transformed these fresh deletion mutant strains and selected for transformants on dual selection SD media plates for Leu- and Ura- conditions at 30 °C. We colony purified transformants prior to determining MSI mutation rates. The MSI rate for individual yeast deletion mutant strains was determined by fluctuation analysis using the method of the median from samples of 15 independent cultures [
Plasmid rescue, PCR and DNA sequencing
FOAR colonies were patched on SD-leu+FOA plates. Plasmids were recovered using methods as previously described [
We would like to thank Angela Chu for providing yeast deletion strains, assistance in confirming the PAU24 deletion mutant and general guidance about using the homozygous diploid deletion pools. This work was supported by the following grants from the NIH: 5K08CA96879-6 to H.P.J., DK56339 to H.P.J, 2P01HG000205 to K.W. and H.P.J.
- Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010 Jun;138(6):2073-2087 e3.
- Jasperson KW, Tuohy TM, Neklason DW, Burt RW. Hereditary and familial colon cancer. Gastroenterology 2010; 138(6): 2044-2058.
- Jun SH, Kim TG, Ban C. DNA mismatch repair system. Classical and fresh roles. FEBS J 2006; 273(8): 1609-1619.
- Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 2002; 418 (6896): 387-391.
- Ji HP, King MC. A functional assay for mutations in tumor suppressor genes caused by mismatch repair deficiency. Human Molecular Genetics 2001; 10(24): 2737-2743.
- Ishioka C, Suzuki T, FitzGerald M, Krainer M, Shimodaira H, Shimada A, et al. Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast. PNAS 1997; 94(6): 2449-2453.
- Boeke JD, LaCroute F, Fink GR. A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Molecular & General Genetics 1984; 197(2): 345-356.
- Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003; 19(2): 185-193.
- Hillenmeyer ME, Fung E, Wildenhain J, Pierce SE, Hoon S, Lee W, et al. The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 2008; 320(5874): 362-365.
- Grant GR, Liu J, Stoeckert CJ, Jr. A practical false discovery rate approach to identifying patterns of differential expression in microarray data. Bioinformatics 2005; 21(11): 2684-2690.
- Lea DA, Coulson CA. The distribution of the numbers of mutants in bacterial populations. Journal of Genetics 1949; 49: 264-285.
- Jeter JM, Kohlmann W, Gruber SB. Genetics of colorectal cancer. Oncology (Williston Park) 2006; 20(3): 269-76; discussion 85-6, 88-89.
- Woods MO, Younghusband HB, Parfrey PS, Gallinger S, McLaughlin J, Dicks E, et al. The genetic basis of colorectal cancer in a population-based incident cohort with a high rate of familial disease. Gut 2010; 59(10): 1369-1377.
- Luo Z, van Vuuren HJ. Functional analyses of PAU genes in Saccharomyces cerevisiae. Microbiology 2009; 155(Pt 12): 4036-4049.
- Rintala E, Toivari M, Pitkanen JP, Wiebe MG, Ruohonen L, Penttila M. Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae. BMC Genomics 2009; 10: 461.
- Engel SR, Balakrishnan R, Binkley G, Christie KR, Costanzo MC, Dwight SS, et al. Saccharomyces Genome Database provides mutant phenotype data. Nucleic Acids Res 2010; 38(Database issue): D433-6.
- Shah SN, Hile SE, Eckert KA. Defective mismatch repair, microsatellite mutation bias, and variability in clinical cancer phenotypes. Cancer Res 2010; 70(2): 431-435.
- Eckert KA, Hile SE. Every microsatellite is different: Intrinsic DNA features dictate mutagenesis of common microsatellites present in the human genome. Mol Carcinog 2009; 48(4): 379-388.
- Hawk JD, Stefanovic L, Boyer JC, Petes TD, Farber RA. Variation in efficiency of DNA mismatch repair at different sites in the yeast genome. Proc Natl Acad Sci U S A 2005; 102(24): 8639-8643.
- Lehner KR, Stone MM, Farber RA, Petes TD. Ninety-six haploid yeast strains with individual disruptions of open reading frames between YOR097C and YOR192C, constructed for the Saccharomyces genome deletion project, have an additional mutation in the mismatch repair gene MSH3. Genetics 2007; 177(3): 1951-1953.
- Gietz RD, Woods RA. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 2002; 350: 87-96.
- Chu AM, Davis RW. High-throughput creation of a whole-genome collection of yeast knockout strains. Methods Mol Biol 2008; 416: 205-220.
- Pierce SE, Davis RW, Nislow C, Giaever G. Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nat Protoc 2007; 2(11): 2958-2974.
- Wierdl M, Dominska M, Petes TD. Microsatellite instability in yeast: dependence on the length of the microsatellite. Genetics 1997; 146(3): 769-779.
- Robzyk K, Kassir Y. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Research 1992; 20(14): 3790.