Insertion of Mutant PA618V into Saccharomyces Cerevisiae and Its Effects on HNPCC

Carrithers B., Carter T., Hill S., and Sims A.Molecular Biology and Genomics

Dr. Anna Powolny

Colorectal cancer (CRC) is on the rise as one of the most common cancers diagnosed in the United

States. Death rates associated with CRC include over 50,000 individuals because of genetic

mutations in DNA mismatch repair (MMR) system genes, MSH2 and MLH 1, which cause cancer.

These genetic mutations are commonly seen in the most inherited type of CRC: hereditary non-

polyposis colorectal cancer (HNPCC). HNPCC is characterized by an aneuploid tumor with

microsatellite instability (MIN); this is where the MSH2 gene in the DNA MMR system is defective.

MSH2 is highly conserved; therefore, in order to detect if a tumor in humans is benign or

malignant, a yeast organism is used as a model. Clinically relevant mutants (plasmids with MSH2

mutants) are used to simulate the mutation in DNA MMR and are placed into the yeast model. The

experiments conducted, which are used to simulate the mutation found in HNPCC, include plate

streaking of E.coli, plasmid isolation, enzyme restriction, agarose gel electrophoresis, polymerase

chain reaction (PCR), 5-FOA complementation assay, and SDS-PAGE and Western Blot to

determine if the inherited MSH2 gene mutation will affect its amount of protein in the yeast model,

its activity, and whether the mutant pA618V would be malignant or benign.

Introduction

Colorectal cancer (CRC) is the 3rd most common inherited cancer diagnosed in America today, which is

accompanied by a death rate of 50,000 individuals in 20031. There are two types of inherited colorectal

cancer: 1) Familiar Adenomatous Polyposis (FAP) and 2) Hereditary Non-Polyposis Colorectal Cancer

(HNPCC). FAP is characterized by an aneuploid tumor with chromosomal instability (CIN) and polyps

located in the distal end of the colon, while HNPCC is characterized by a diploid tumor with

microsatellite instability (MIN), no polyps, and located in the proximal end of the colon1,4. Hereditary

Non-Polyposis Colorectal Cancer (HNPCC), also know as Lynch Syndrome, is a colorectal cancer that is

the most common of colorectal cancers and accounts for about 10 percent of diagnoses each year1.

HNPCC was first discovered by Alfred Warthin in 19131, and it is derived from defects found in DNA

mismatch repair (MMR) genes1. The cause of these defects is errors within DNA replication. Without an

intact mismatch repair system, DNA begins to accumulate mutations2. Repetitive DNA (Microsatellite

DNA) that is highly unstable in MMR defective strains are hallmarks for HNPCC1. In a mismatch repair

system, there are two clinically relevant homologs that are used: MutS and MutL1. In these homologs,

there are several types of genes that are found in the DNA MMR system of both prokaryotes and

eukaryotes. Most of the mutations in the DNA MMR system that cause colorectal cancer include MSH2,

MSH3, MSH6 MLH1, PMS1, and PMS2, with MSH2 and MLH1 accounting for most HNPCC cases2.

From this background information, the hypothesis was formed to determine if the inherited MSH2 gene

mutation will affect its amount of protein in the yeast model and its activity. The tests and activities

completed include plate streaking of E.coli, plasmid isolation, enzyme restriction, agarose gel

electrophoresis, polymerase chain reaction (PCR), 5-FOA complementation assay, and SDS-PAGE and

Western Blot.

Materials and Methods

Bacteria Streaking: While wearing gloves, a pre-sterilized inoculating loop was taken and held under the

Bunsen burner flame at 45 degrees for a few seconds. The E. coli sample was opened and the loop was

then dipped into the sample. The culture lid was removed away from the face. Then, the inoculation loop

containing the E. coli sample was spread on the culture plate using a zigzag motion. These steps were

repeated three more times in three sections on the plate. The top was placed back over the culture and was

incubated for 48 hours and 20 degrees Celsius.

Plasmid Isolation: Three tubes, which contained a bacterial culture, were labeled pMSH2 (wild-type),

pRS413 (vector), and pA618V (mutant) and were centrifuged to collect bacterial cells at 4000 RPM for 5

minutes. The supernatant in each tube was decanted. To remove excess media, tubes were turned upside

down for 3 minutes on a paper towel to drain. 300µL of Cell Resuspension Solution was pipetted into

each tube to resuspend the pellet. 300µL of Cell Lysis Solution was pipetted into each tube and was

mixed by inverting four times. The suspended pellet was transferred into a new tube and 300µL of

Neutralization Solution was added to each tube. Then, the tubes were mixed by inverting four times and

were placed spun in the micro centrifuge at 14000 RPM for 10 minutes and were placed on ice.

Luer-Lock filtration mini columns were assembled and labeled pMSH2, pRS413, and pA618V.

The columns were placed into vacuum manifolds. 1 mL of Wizard MiniPrep DNA Purification Resin and

the supernatant from each tube were pipetted into their respective filtration columns, and the mixture was

pushed into the mini column with a plunger. 2 mL of Wash Solution was pipetted into each column and

was pushed through with a plunger. The mini columns were disassembled from their barrels and were

transferred to labeled 1.5 ml microcentrifuge tubes and were centrifuged for 2 minutes at 14000 RPM. 50

µL of 1x Tris was added to the mini-columns and was centrifuged at 14000 RPM for 1 minute, causing

the plasmid DNA to be collected into the microcentrifuge tubes. The tubes were capped and stored in a -

20 degree Celsius freezer.

Enzyme Restriction: While the plasmid DNA thawed out on ice, a master uncut mix was made using 8

µL buffer, 48 µL H2O, and 4 µL BSA. A master cut mix was made using 8 µL buffer #4, 48 µL H2O, 4

µL BSA, and 4 µL of Hha1 enzyme. Six microcentrifuge tubes were labeled: uncut pMSH2, cut pMSH2,

uncut pRS413, cut pRS413, uncut pA618V, and cut pA618V. The cut tubes will have the restriction

enzyme that will cut the plasmid, the uncut will not. In each labeled tube, 5 µL of the assigned plasmid

was inserted along with 15 µL of the cut/uncut mix. The microcentrifuge tubes were centrifuged for a few

seconds and incubated in a 37 degrees Celsius water bath for 50 minutes. The tubes were then stored in a

-20 degree Celsius freezer.

Agarose Gel: The gel rack was assembled with the comb into the electrophoresis rig. Molten 1% agarose

gel pre-stained with Ethidium bromide was poured into the gel rack and cooled for 20 minutes. The rack

was inverted, the comb was removed, and 1x TBE buffer was added over the gel. 10 µL of the 1 Kb DNA

ladder standard was pipetted into the first well, and the cut and uncut plasmid mixtures into the next six

wells with 2 µL of loading dye. The lid was placed over the rig and the electrodes were connected. The

rig was plugged into the power supply and set to 120 mV. The gel was run for 50 minutes and was taken

into the lab to get photographed using the GBox iChemi XR Genesys.

PCR Reaction: A serial dilution was made to dilute the solutions. 2 µL of extracted plasmid was taken

and added to 98 µL of water. Then 2 µL of that solution was added to 38 µL of water for a 1000x

dilution. Three tubes containing Polymerase Chain Reaction beads, (which are composed of stabilizers,

dNTPs, Taq DNA polymerase, reaction buffer, Tris HCl, KCl, and MgCl2) were labeled vector, wildtype,

and mutant. In the vector tube containing the PCR bead, 2 µL of primer, forward and reverse were

combined, 3.8 µL of the vector, and 19.2 µL of water was added for a final volume of 25 µL. In the

wildtype tube containing the PCR bead, 2 µL of primer, 2.3 µL of the wildtype, and 20.7 µL of water was

added for a final volume of 25 µL. In the mutant tube containing the PCR bead, 2 µL of primer, 2.3 µL of

the mutant, and 20.7 µL of water were added for a final volume of 25 µL. The tubes were placed in a Bio-

Rad thermocycler and the PCR process began. The samples incubated at 95 degrees Celsius for 5 minutes

to activate the Taq Polymerase, then one minute cycles of 45 degrees Celsius to anneal, 72 degrees

Celsius to elongate, and 95 degrees Celsius to elongate were repeated 34 times for a total of 35 one

minute cycles. The last elongation phase lasted for 10 minutes then the solutions were placed at 4 degrees

Celsius for 2 hours to cold soak. After the cold soak, the tubes were placed and stored in a -20 degree

Celsius freezer for 5 days.

Restriction Enzyme: 8 microcentrifuge tubes were labeled: pMSH2 cut, pA618V cut, pRS413 cut,

pMSH2 uncut, pA618V uncut, pRS413 uncut, Uncut mix, and Cut mix. To the cut mix tube, 4 µL of

enzyme HhaI and 4 µL of buffer #4 were added and spun in the microcentrifuge. To the uncut mix, tube

4µL of H2O and 4 µL of buffer #4 were added and spun in the microcentrifuge. 8 µL of PCR sample was

added along with 2 µL of cut mix to the pMSH2 cut tube, the pA618V cut tube, and the pRS413 cut tube.

8 µL of PCR sample was added along with 2 µL of the uncut mix to the pMSH2 uncut tube, the pA618V

uncut tube, and the pRS413 uncut tube. The six tubes were mixed and spun in the microcentrigue for a

few seconds then placed in the incubator at 37 degrees Celsius for 50 minutes. After the samples were

incubated, 2 µL of 6x loading buffer was added to each tube to prepare it for agarose gel electrophoresis.

Agarose Gel: The gel rack was assembled with the comb into the electrophoresis rig. Molten 2% agarose

gel pre-stained with Ethidium bromide was poured into the gel rack and cooled for 20 minutes. The rack

was inverted, the comb was removed, and 1x TBE buffer was added over the gel. 12 µL of the 100 bp

DNA ladder standard was pipetted into the first well, and the cut and uncut plasmid mixtures into the next

six wells. The lid was placed over the rig and the electrodes were connected. The rig was plugged into the

power supply and set to 120 mV. The gel was run for until it was 2/3 of the way down the gel and was

taken into the lab to get photographed using the GBox iChemi XR Genesys.

Yeast Transformation:

A 10 mL yeast sample containing no MSH2 and no ability to make its own histadine or tryptophan was

centrifuged for one minute at 10000 rpm. The supernatant was decanted and was resuspended in 750 µL

of sterile water and was centrifuged again. The supernatant was drained and the remaining cell pellet was

resuspended in 400 µL of plate solution. 0.5mL of suspended yeast, 20 µL 1M DDT, 10 µL boiled carrier

DNA, and 10 µL plasmid DNA was added into each tube. The contents were mixed and incubated for 30

minutes at 30 degrees Celsius. The cells were then heat shocked for 30 minutes at 42 degrees Celsius. The

mixture was centrifuged for one minute at 10000 rpm. The supernatant was removed and the pellet was

resuspended in 100 µL of sterile water. The entire volume of cells was placed on the surface of an SD-trp-

his plate and was spread evenly across the plate using inoculating loops.

FOA and preparation:

Empty tubes were obtained and labeled 1.0 pMSH2, 0.1 pMSH2, 0.01 pMSH2, 1.0 pRS413, 0.1 pRS413,

0.01 pRS413, 1.0 pA618V, 0.1 pA618V, and 0.01 pA618V. The transformed yeast cells were spun at

4000 rpm for 5 minutes and resuspended in 500µL of water, and were then placed into their

corresponding 1.0 labeled tubes. A serial dilutions was made for the 0.1 tubes, 50µL of the 1.0 yeast cells

was added to 450µL of water. For the 0.01 dilutions, 50µL of the 0.1 dilution was added to 450µL of

water. A square was drawn on an FOA plate and was divided into nine sections: 1.0 pMSH2, 0.1 pMSH2,

0.01 pMSH2, 1.0 pRS413, 0.1 pRS413, 0.01 pRS413, 1.0 pA618V, 0.1 pA618V, and 0.01 pA618V. 75

µL of each dilution was pipetted into the center of each corresponding square. The plates were flipped

after an hour so the transformed yeast would have time to absorb into the plate.

Yeast Protein Extraction:

100 µL of each of the cells were centrifuged at 13000 rpm for one minute. The cell pellets were then

resuspended in 100µL alkaline lysis buffer containing 0.1M NaOH, 0.05M EDTA, 2% SDS and 2%

BME. The mixtures were heated on a heating block at 90 degrees Celsius for ten minutes. Then the

solutions were neutralized by adding 4µL of 4M acetic acid. The samples were heated again for ten

minutes at 90 degrees Celsius. Then the samples were spun for five minutes at 14000 rpm. The

supernatant was transferred into fresh tubes and the samples were prepared for gel. 20 µL of sample

buffer was added to 20µL of protein extract. 30µL of the prepared samples were then loaded onto the

SDS-PAGE gel.

SDS Page and Western Blot:

3 µL of standard of pre-stained standard was placed into the first well and 30 µL of each sample was

loaded into the following wells. The gel was run for one hour at 120 V. Afterwards, the gel was

transferred to a nitrocellulose membrane using methanol and a transfer buffer that lubricated the sponge,

filter paper, membrane, and gel used in the transfer stack. The transfer was run at 200 milliamps for one

hour. The membrane was blocked for one hour at room temperature with 5% milk blocking solution. The

membrane was incubated with a 1:2000 diluted primary Anti Hemagglutinin rabbit polyclonal antibody at

4 degrees Celsius overnight. The membrane was then washed with TBST, three times for five minutes

each. The membrane was incubated with a secondary Horseradish Peroxidase HA tagged polyclonal

antibody at room temperature for one hour, and then washed with TBST, which contains Tris-Buffered

Saline and Tween 20. The membrane was developed using chemiluminesence.

Results

Streaking Bacteria

The purpose of streaking bacteria was to obtain isolated colonies of E.Coli bacteria. By spreading a large

amount of bacteria over the large surface area of the plate, the amount of bacteria was diluted until

individual cells were placed on the surface of the plate. From observation, single colonies of E.coli

bacteria arose and were used as cloning.

Figure 1. Agar plate of E. Coli bacteria, small colonies of bacteria grown.

Isolating Plasmid DNA from Host Bacterial Strains

During plasmid DNA isolation, cell lysis occurred to eliminate intracellular macromolecules allowing for

the Plasmid DNA to be more prominent. While mixing Cell Lysis Solution in each tube, the solutions

appeared clear and viscous. The formation of precipitation containing cellular debris and chromosomal

DNA was observed. As Shown in table 1, the concentration of the plasmid DNA extraction for pRS413

was 131.3 ng/uL, pMSH2 was 217.7 ng/uL, and the mutant pA618V was 218.0 ng/uL. The ratios for

pMSH2 and pA618V showed that there was no protein present in the extractions.

Table 1. Ratio of amount of Plasmid DNA in various mutant types.

Restriction Enzyme Digestion of Plasmid DNA and

Agarose Gel electrophoresis

Restriction enzymes are able to scan along DNA searching for various sequences of bases. The restriction

enzyme attaches to the DNA and cuts into each strand of the double helix and cuts the molecule into

fragments. Agarose gel electrophoresis was used to resolve the mixtures of the cut plasmid DNA. The

fragments were visually identified by the use of the UV transilluminator. Fading was seen at the lower

end of the photographic image, indicating that gel should have been ran for a slightly longer period of

Mutant Type Ratio Concentration

PRS413 1.7 131.3 ng/ul

PMSH2 1.88 217.7 ng/ul

PA618V 1.87 218.0 ng/ul

time. Sharp bands were observed, indicating that there was little to no protein present in plasmid samples.

There is evidence of supercoiling, represented by bands being located at the top. Based upon the

photographic image, it was concluded that digestion worked and that the mutant (pA618V) was different

than the wild type (pMSH2). As seen in Figure 2, the digestion was successful because the cut bands

migrated further than the uncut bands. Cut pA618V is distinguished from the cut wild-type (pMSH2)

band by the difference in location of the restriction enzyme site, showing that there is a mutation present

in the cut pA618V.

Figure 2. DNA bands after restriction enzyme cutting of plasmids present in 1% agarose gel

electrophoresis. Bands are seen through UV transilluminator and compared to the 1 Kb ladder standard,

located at the far left of the gel. Bands migrate due to their sizes. Smaller fragments are able to migrate

farther and faster than bigger fragments.

18 18 24 30 46 420

1,000

2,000

3,000

4,000

5,000

6,000

7,000

Standard Curve for Restriction Enzyme Gel Elec-trophoresis

Distance Traveled (in mm)Fragment Size (in bps)

Distance Traveled (in mm)

Frag

men

t Size

in b

ps)

pA618V pA618VpRS413pMSh2 pMSh2 pRS413

Figure 3. The standard curve, the 1 Kb ladder, is used to estimate the band sizes of DNA after restriction

enzyme cutting of plasmids and agarose gel electrophoresis (The uncut fragments are first, then the cut

fragments). Estimation of band sizes is as followed: cut pMSH2 (750-1,000 bps), cut pRS413 (250 bps),

cut pA618V (2,000-3,000 bps), uncut pMSH2 (6,000 bps), uncut pRS413 (3,000 bps), and uncut pA618V

(6,000 bps).

Polymerase Chain Reaction

In polymerase chain reaction, the mutant pA618V was amplified from a single copy of the target DNA

segment, MSH2. Based on our results, no bands were anticipated to appear in the vector, pRS413, but due

to contamination, bands are slightly visible. In the mutant, pA618V, the cut and uncut bands are located

in the same area, not distinguishing the bands from one another. The pMSH2 cut bands migrated down in

two fragments. The difference between the cut mutant and the cut wild type is the size of the gene. The

PCR gel shows the mutation that is present in the amplified mutant pA618V’s MSH2 gene is larger than

the amplified wild type pMSH2’s gene, indicating the difference in the gene sizes.

Figure 3. DNA bands after PCR in 2% agarose gel. Cut mutant, pA618V, is cut higher than the wild-type, pMSH2, showing that the mutant, pA618V, is different from the wild-type, pMSH2.

Yeast transformation

Yeast was plated with reporter plasmids that restored their ability to make Histidine and Tryptophan

allowing for the yeast colonies to arise. Based on the photographic images, there are no conclusive results

on MSH2; only transformation took place.

1) pMSH2 2) pRS413 3) pA618V

Complementation Assay

In this yeast transformation, the mutant pA618V was studied to determine the effects of pMSH2, wild-

type deletion. No exact results were confirmed due to discrepancies in determining whether the pRS413

and pMSH2 had been switched during labeling processes. The anticipating results would have included:

NO growth in the wild-type pMSH2, abundant growth in the vector pRS413, and from there, determine

Figure 4. The photographic image of yeast transformation after the restoration of the ability to make Histidine and Tryptophan.

whether mismatch repair had been restored in the mutant pA618V. Due to mislabeling, the wild-type

pMSH2 and vector pRS413 were switched. Our results now show growth in wild-type pMSH2, some

growth in vector pRS413, and abundant growth in our mutant pA618V. From the observations from the

5-FOA plating, it was concluded that out mutant, pA618V, was malignant as the DNA mismatch repair

system was not restored, allowing colonies to grow continuously with mutations present.

Figure 3. Complementation assay proves that mutant A618V produced more colonies than wild-type

pMSH2, therefore MMR system did not repair functionality.

Discussion

There are various mutations that possibly contribute to defects in the MSH2 gene causing HNPCC. In the

Characterization of Pathogenic hMSH2 Missense Alleles, fifty-four missense mutations were introduced

to the yeast, MSH2, and tested for mismatch repair on a molecular level. “The missense mutation is

introduced to the cognate yeast MSH2 coding sequence so that the MSH2 gene harboring the mutation is

distinguishable from the wild type by restriction endonuclease digestion”(Gammie 1, 35)1. Based upon

the results of the Gammie 1 report, the PCR amplification allowed for identification of the mutagenized

plasmids. Much like the Gammie 1 report, the confirmation of the mutation by Restriction Digestion and

PCR in this presented research, was confirmed by the same analysis of patterns in band sizes and

locations to the wild-types and mutated plasmids. In the Gammie mismatch repair assays, the

mutagenized plasmid, the pMSH2, and the PRS413 vector were transformed using the yeast reporter

strain AGY75 and the colonies that were observed from the tranformations were then tested for DNA

mismatch repair. In the immunoblotting, the secondary antibody used was horseradish peroxidase much

like what was used in the experimental design presented in this research. In the Characterization of

Pathogenic hMSH2 Missense Alleles, detrimental substitutions altered the levels of MSH2 contributing to

the stability of the form and function of MutS (Gammie 2, 715)5. In the research presented in this report,

mismatch repair was defective in the mutant, pA618V, on the 5-FOA plate, determining the mutation as

malignant. In the Characterization of Pathogenic hMSH2 Missense Alleles, “cells displayed an elevated

rate of resistance to 5-FOA, which was indicative of instability”. Overall, most of the missense mutations

in the Gammie report, except the pseudo-wild-type variants, significantly altered Msh2 functioning and

MMR. The characterization of mutations within mismatch repair allotted for understanding of the cellular

and molecular defects of the MSH2 gene and its clinical relevance to HNPCC.

Conclusion

The data presented in this report characterize an extensive set of experiments to determine mutations in

the mismatch repair gene, MSH2. We were able to distinguish the functionality of the mutation and

determine if the mutation significantly altered the functioning of the MSH2 gene. The research presented

can conclude that the absence of MSH2 can alter the functionality of mismatch repair and cause defects in

humans. Genetic testing for these human defects, such as HNPCC, is primarily determined by the

presence of MSH2. Defective mismatch repair is the inability to repair mismatches, small deletions and

insertions, which were tested in these experiments. Our results so far, do prove that the deletion and

mutation of MSH2 has affected the yeast cell and its ability to rep air, but due to complications in the

complementation assay no conclusions can be drawn and are still under study. Anticipating results back

from the mislabeling of the complementation assay plate, the prediction would be that pA618V would be

malignant, as growth is seen on the 5-FOA complementation assay.

References

1. Gammie, Alison E., and Naz Erdeniz. "Characterization of Pathogenic Human MSH2 Missense Mutations Using

Yeast as a Model System: A Laboratory Course in Molecular Biology." Cell Biology Education 3 (2003): 31-48.

Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014. Web. 01 May 2013.

2. "Hereditary Nonpolyposis Colon Cancer (HNPCC)." - Information About Cancer. Stanford Medicine, n.d. Web.

20 Feb. 2013.

3. "Lynch Syndrome." Genetics Home Reference. Apr. 2008. Web. 20 Feb. 2013.

4. Alberts, Stephen R., Deborah Citrin, and Miguel Rodriguez-Bigas. "Colon, Rectal, and Anal Cancers." - Cancer

Network. UBM Medica LLC, 8 Mar. 2013. Web. 21 Mar. 2013.

5. Gammie, Alison E., Naz Erdeniz, Julia Beaver, Barbara Delvin, Afshan Nanji, and Mark D. Rose. "Functional

Characterization of Pathogenic Human MSH2 Missense Mutations in Saccharomyces Cerevisiae." Department of

Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014 (2007): 707-21. Web. 01 May

2013.