examination of the apn2 homolog in tetrahymena...

BMS110 Honors, MSU Fall 2011

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Examination of the APN2 homolog in Tetrahymena thermophila

Krysta Baker and Kelly Perkins

Fall 2011

Abstract

AP endonuclease enzymes play a crucial role in repairing DNA damage, often through a pathway

called base excision repair. The BER pathway repairs damage done to DNA in the cell by either

harmful agents within the cell or outside factors such as anti-cancer drugs (Al-Attar et al., 2010).

In cancer cells, however, repairing damage done to DNA by anti-cancer drugs can increase the life

of these deadly cancer cells. APN2 is part of the AP endonuclease family of enzymes that partakes

in this function. Through research, evidence is showing that by inhibiting these enzymes in cancer

cells and allowing the cell to undergo apoptosis and die instead of repairing the harmful genetic

code, the effectiveness of chemotherapy and other therapeutic methods to eliminate cancer could

likely increase (Jiang et al., 2010). In order to learn more about these enzymes and their processes,

a homolog of APN2 in Tetrahymena thermophila was found by use of bioinformatics tools.

Tetrahymena thermophila is an ideal organism for study because it is a single-celled organism

with a fast reproduction rate. After cloning this gene and transforming it into a plasmid, it can be

stored until more research can be conducted. Continued investigation in the functions of APN2 and

other AP endonuclease enzymes, especially of the enzymes in human cells, could lead to

breakthroughs in the medical world of cancer treatment.

Introduction

Both APE1 and APN2 belong to a family of endonuclease enzymes. APE1 is of the strain

Trypanosoma cruzi, a parasitic organism, while APN2 is the homolog gene in Saccharomyces

cerevisiae, a specific strain of yeast. For simplicity and because both genes perform similar


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functions in their respective organisms, the name APN2 will be used throughout this study to

encompass both. These enzymes accounts for 95% of all apurinic/apyrimidinic (AP) endonuclease

activity in a cell and is vital in DNA base excision repair. This pathway, known as the BER

pathway, is vital in repairing mutations and changes in DNA that are often caused by endogenous

or exogenous agents. This damage can lead to AP sites that are potentially harmful to the genetic

composition of the cell, and APN2 is most often the target enzyme to repair these lesions (Al-Attar

et al., 2010). In certain cases, many of the DNA lesions that undergo the BER pathway are caused

by cancer treatment or anti-cancer drugs (Ma, 2008). In such situations, APN2 can potentially be

hindering the effectiveness of these anti-cancer drugs. The anti-cancer drugs seek to damage the

cancer cell’s DNA so that the cell will undergo apoptosis--otherwise known as programmed cell

death--in which the cell basically kills itself. By attempting to repair the damage these anti-cancer

drugs are doing to the cancer cells, APN2 is increasing the lives of the harmful cancerous cells

(Jiang et al., 2010). In fact, high levels of the AP endonuclease enzymes may have a link to

resistance of chemotherapy and radiation in the treatment of cancers such as breast cancer, ovarian

cancer, gastroesophageal cancer, and pancreatico-biliary cancer (Bapat, 2009; Al-Attar et al.,

2010). By potentially finding inhibitors to the AP endonuclease enzymes in human cells, the

ability for anti-cancer treatments to be effective may likely increase. Further investigation into the

functions of these enzymes is needed in order to determine whether a link to cancer treatment

resistance exists.

Through the use of bioinformatics tools, we were able to find a homolog of APN2/APE1 in

Tetrahymena thermophila. After isolating this gene for Tetrahymena cells, the use of primers

allowed us to copy the specific gene millions of times through polymerase chain reactions (PCR).

Agarose gel electrophoresis was used to determine whether the gene was successfully copied

through PCR, and then the gene was transformed into a plasmid vector for storage in E. coli


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bacteria. A computer-generated plasmid map was constructed as a tool for visually seeing what the

plasmid will look like with the cloned gene inside it and will be a valuable tool to future

researchers who receive this plasmid to study. Restriction enzyme digests were created in order to

verify that the plasmid contains the specific cloned gene. Confirmation by agarose gel

electrophoresis showed that the gene was successfully inserted into the plasmid vector of E. coli,

and the samples chosen were cryopreserved in glycerol to keep frozen until further

experimentation on the functions of the gene and possible link to cancer can be performed.

Materials and Methods

Bioinformatics and Primer Design

[See “Lab 3: Bioinformatics & Molecular Computational Tools” for complete protocol (Smith,

2011)]

In order to identify a homolog of APE1 in Tetrahymena thermophila, bioinformatics information

was utilized. The first step was to identify the amino acid sequence of APE1 through the use of the

protein database on the NCBI website, http://www.ncbi.nlm.nih.gov/. The starts and stops of the

gene were found. A homolog in Tetrahymena of APE1 was then found in the Tetrahymena

Genome Database Wiki, http://www.ciliate.org/, using the BLAST search tool and the found

amino acid sequence. The same Tetrahymena Genome Database was used to find the protein

sequence, nucleotide sequence, genomic sequence, and ESTs of the homolog of APE1. The

homolog’s genomic and coding sequences were compared using the MGAlignIt website,

http://proline.bic.nus.edu.sg/mgalign/mgalignit.html. A protein sequence comparison was also

performed using the BLAST program within the NCBI site. A specialized BLAST was also used

to compare protein sequences (BL2SEQ). All comparisons were used to ensure the proper

homolog was identified to APE1 in Tetrahymena. The gene APE1 is now called APN2 because it


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is a homolog in the strain Saccharomyces cerevisiae and has a more convenient name for this new

research. The genomic sequence with underlined ESTs can be seen in Figure 1.

The bioinformatics data was then used to create primers necessary for further experimentation of

the specific gene. The data collected was sent to the Integrated DNA Technologies laboratory and

both forward and backward primers were found. The primers were designed for the 5’ and 3’

regions of the APE1 gene. The primers designed can be seen in the table below. Primers are

necessary to amplify the specific gene in the process of polymerase chain reaction (PCR).

Primer Type Sequence Amount

(nMoles)

Primer Annealing

Temperature (Tm)

Forward Primer 5’-CAC CCT CGA GAT TAA ATC

TTT TAG ATT TTC AGC TTC -3’ 36.4 58.9°C

New Forward Primer (for new possible start)

5’- CAC CCT CGA GAG TAA AAT

AAT TAA AAA ATC AAG CTC -3’ 38.8 57.9°C

Reverse Primer 5’- AGA GCC TAG GTC AAT TAG

ATT TAT TTG TTT ATG TAT TT -3’ 33.9 57.0°C

Tetrahymena Genomic DNA Isolation and Quantification

[See “Lab 4: Tetrahymena Genomic DNA Isolation” for complete protocol (Smith, 2011)]

The isolation of the Tetrahymena genomic DNA was necessary to use the DNA as a template in

the next PCR process. The procedure started with Tetrahymena culture and the cells were

collected through centrifuging. Urea Lysis Buffer was added to break open the Tetrahymena cells

and release their DNA, and then phenol extraction was done by the use of

phenol:chloroform:isoamyl alcohol. Sodium chloride was added to the extracted lysate to reduce

the carbohydrate content of the final precipitate. An equal volume of isopropyl alcohol was added

to the lysate (which was approximately 700 µL), and then was centrifuged and the supernatant

poured off. Ethanol was added to solubilize the salts and remove them from the pellet formed.


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This was again centrifuged, the supernatant poured off, and then pellet was allowed to dry

completely. A TE buffer and RNase A was added. This entire process isolated the Tetrahymena

DNA from the original Tetrahymena culture.

DNA Quantification

[See “Lab 4: Tetrahymena Genomic DNA Isolation” part II for complete protocol (Smith, 2011)]

To quantify the amount of DNA in the solution, a NANODROP 2000 machine was used. This

machine, called a spectrophotometer, uses wavelengths of 260 nm and 280 nm, and a ratio of these

two numbers provides an estimated purity of the DNA. After the NANODROP 2000 was blanked

with water, 1 µL of the isolated Tetrahymena DNA was placed on the spectrophotometer. The

spectrophotometer displayed the concentration of the DNA and the absorbance readings at 260nm

and 280nm—A260 and A280 respectively. Dividing A260 by A280 gave the estimated purity. Because

both my lab partner and I were able to successfully isolate separate DNA samples, we were able to

select which sample we wanted to continue experimenting on. We based our decision on the

highest concentration level, and these results can be seen in Table 1.

Polymerase Chain Reaction (PCR)

[See “Lab 5: Polymerase Chain Reaction (PCR)” for complete protocol (Smith, 2011)]

Polymerase chain reaction was used to copy the specific APN2 gene in the isolated Tetrahymena

DNA millions of times by use of the primers found from the bioinformatics data. The three main

steps of PCR are denaturation of the template DNA, annealing of the primers to template DNA,

and extension of the primer (DNA synthesis). The PCR reactions contained either 3 µL genomic

DNA or 3 µL cDNA (1:10 dilution) depending on which stock, 1.5 µL forward primer, 1.5 µL

reverse primer, 1.5 µL Phusion polymerase, 30 µL GC buffer, 30 µL Betaine, 3 µL dNTPs, and


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79.5 µL sterile distilled water. After the working stock was created and the annealing temperatures

were calculated, a thermocycler was used to perform the PCR. The steps of the thermocycler with

our specific temperatures began with heating the reactions for 1 minute at 98°C to denature

genomic DNA. Next, 34 cycles of: 20 seconds at 98°C (denaturation), 25 seconds at primer

annealing temperature (which included temperature #1 (G1, C1, CN1) at 53.7°C; temperature #2

(G2, C2, CN2) at 59.4°C; and temperature #3 (G3, C3, CN3) at 60.5°C), and 1.5 minutes of

polymerase extension at 72°C. Next, the reaction was held for 10 minutes at 72°C and finally held

at a constant temperature of 4°C. G, C, and CN refer to genomic DNA, cDNA, and the cDNA with

a primer that corresponds to the potentially new start of the APN2 gene as mentioned in the

bioinformatics, which is later proven to be the incorrect start anyway. The copies generated

through PCR were analyzed and identified with agarose gel electrophoresis.

Agarose Gel Electrophoresis

[See “Lab 6: Agarose Gel Electrophoresis” for complete protocol (Smith, 2011)]

Electrophoresis was used to separate, identify, and purify DNA fragments. Agarose gel

electrophoresis utilizes a combination of electrical and frictional forces to separate the DNA, and

the location of the DNA on the gel can be visualized by direct staining of the DNA with dyes such

as the ones used in this experiment—xylene cyanol-blue and bromphenol blue-purple. A 1.6%

agarose gel was used to hold our samples. 10 µL of each of our nine samples after PCR were

mixed with 1 µL of 10X sample dye and loaded into the wells of the gel. The loading pattern was

determined by the primer annealing temperatures from the polymerase chain reactions. After the

lab procedure was completed and the gel had run until the bromphenol blue band was ¾ the way

down the gel, a picture of the gel was taken by a UV light box. Later, Dr. Joshua J. Smith, our

professor, redid every groups’ PCR and gel electrophoresis labs in order to be sure the best


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possible results were found. These gel pictures, as seen in Figure 2, utilize a 1 kb ladder to

determine the size of each sample run on the gel. The gels were analyzed to verify if the PCR was

successful in copying the gene based upon the sizes predicted in the bioinformatics.

Cloning and Transformation

[See “Lab 7c: TOPO Cloning and E. coli Transformation” for complete protocol (Smith, 2011)]

This part of the research entailed cloning the PCR-copied gene into the pENTR/D-TOPO vector

plasmid by use of a pre-made kit. The necessary components of the reaction included the .75 µL of

PCR product itself, 1 µL salt solution, 3.25 µL sterile water, and the 1 µL TOPO vector to bring

the final volume of the mix to 6 µL. This TOPO reaction solution was then introduced to E. coli

cells and joined via a TOPO vector plasmid carrier. After the plasmid was cloned into E. coli, the

reaction was spread onto LB plates to incubate and be analyzed to visually see if cloning of APN2

homolog of Tetrahymena into E. coli was successful. The results from this will be seen in a later

lab.

Plasmid Map Construction and Enzyme Digestion Design

[See “Lab 8: Construction of Plasmid Map & Restriction Enzyme Digestion Design” for complete

protocol (Smith, 2011)]

Assuming that the cloning of the APN2 gene of Tetrahymena thermophila was successful into E.

coli, a plasmid map was created and will be a vital tool for future scientists to visually see a

representation of the plasmid they will be experimenting on. A plasmid map of the plasmid cloned

into E. coli was designed using the Gene Construction Kit 3.0. Restriction enzymes were also

indentified on the plasmid that will help to confirm that the APN2 homolog gene is actually inside

the plasmid and was successfully cloned. The APN2 cDNA plasmid map generated can be seen in


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Figure 3. This gel shows the number of bands and band sizes created by the restriction enzymes

that are introduced to the target plasmids. If the gene was successfully cloned and implemented

into the plasmid, the bands on the gel picture will be the same as the computer model. Digest 1

was predicted to have 3 bands at 266, 844, and 2,674 bases. Digest 2 was predicted to have 2

bands at 1,194 and 2,590 bases.

Plasmid Purification and Restriction Enzyme Digest

[See “Lab 10: Plasmid Purification & Restriction Enzyme Digest” for complete protocol (Smith,

2011)]

In order to confirm that the correct gene was inserted into the plasmid vector, the plasmid first had

to be isolated. By placing six samples of the E. coli plasmids on a LB-Kanamycin plate and in LB-

Kanamycin liquid media, the plasmids containing the cloned gene with the kanamycin-resistant

gene, as shown in pink on Figure 3, remained and thus were purified out of the sample. The six

colonies all produced growth after incubation for one day. The six samples were then made into

two separate restriction enzyme digests. Digest 1 was composed of 14 µL of NE Buffer 4, 1.4 µL

of BSA, 96.6 µL of water, and 3.5 µL each of the restriction enzymes NheI and EcoRI. Digest 2

was composed of 14 µL of NE Buffer 2, 1.4 µL of BSA, 96.6 µL of water, and 3.5 µL each of the

restriction enzymes XhoI and AvrII. After the digests were created, 17 µL of each of sample was

placed in a microcentrifuge tube along with 3 µL of the purified plasmids. Because six colonies

were chosen and each colony sample was added to each digest, twelve total microcentrifuge tubes

were used. These samples were incubated for at least one hour and then underwent agarose gel

electrophoresis to determine whether the plasmid vectors held the cloned gene. Reference Lab 6

for complete protocol of agarose gel electrophoresis (Smith, 2011).


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Results

Using bioinformatics, a single homolog of the original gene APE1 in Trypanosoma cruzi was

found in Tetrahymena thermophila. The top hit for a homolog in Tetrahymena thermophila was

TTHERM_00794600 with an e-value of 2.4e-21. Because of an extra underlined amino acid in the

APE1 sequence, another possible start of the specific gene was considered. The original coding

sequence of APE1 was 1,191 bases, but the new start shrunk the sequence to1,044 bases. Both

starts were tested on until the right start was predicted in Lab 6, which turned out to be the

originally predicted start found through the NCBI site. Due to conflicts with the name APE1,

APN2 is now the name used—APN2 is the homolog of Saccharomyces cerevisiae.

Lab 4, Tetrahymena Genomic DNA Isolation, gave both my lab partner and I a viable sample of

Tetrahymena cells to use in the subsequent labs. By having more than one sample, we were able to

quantify both samples in the NANODROP 2000 and were able to choose which sample we

thought would be more successful for the research. While both samples produced an A260/A280

greater than 2.0--which indicates a rather pure sample--we chose the tube that had both a higher

concentration and slightly higher A260/A280 purification value, which was labeled as “Tube 2” in

Table 1.

In order to find the primer annealing temperatures in Lab 5 for the polymerase chain reactions, a

specific formula had to be used. The formula was to add the number of adenines and the number

of thymine together from one of the primers and multiply this number by two. Then add the

number of guanines and cytosine together from the same primer and multiply by three. Add the

previous two numbers together to get a primer annealing temperature of either the forward primer

or reverse primer, depending on which bases you were adding. Add together the annealing

temperatures of both the forward and reverse primers and divide by two in order to find the

average primer annealing temperature. While these equations were used as a starting point in Lab


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5, the specific temperatures could not be used in the gradient thermocycler because other

polymerase chain reactions were happening and the temperatures needed were already taken, so

other temperatures had to be used. G1, C1, and CN1 were ran at a temperature of 53.7°C; G2, C2,

and CN2 were ran at 59.4°C; and finally G3, C3, and CN3 were ran at 60.5°C. G2, C2, CN2, G3,

C3, and CN3 were all ran at temperatures set above the Tm (primer annealing temperature) as

specified by the Integrated DNA Technologies lab that constructed the primers, which could be

why these samples did not successfully copy and show in the agarose gel picture. Out of the nine

samples run-- G1, C1, CN1, G2, C2, CN2, G3, C3, and CN3 from PCR—only G1 and C1 showed

on the gel picture at all. Both of these gels were ran at a primer annealing temperature of 53.7°C,

which was below the given Tm. Although CN1 was ran at the same temperature, this sample was

created to test which start was correct from the bioinformatics data, and because only C1 was

shown in the gel, the original start from the NCBI website must be the correct start. G1 appeared

to be predicted correctly at around 1.4-1.5 kb, with the correct size being 1,478 bases. C1 had

extra bands present in the gel--which could have been the presence of primer dimmers-- and was

also shown to have been predicted significantly larger than found in bioinformatics—the gel

showed the C1 band to be around 1.3 kilobases when the correct size is 1,191 bases. Based on this

information, G1 would have been the best sample to continue on with in the research; however,

Dr. Smith noticed that most of the class was unsuccessful with the cDNA, so he redid everyone’s

PCR and agarose gel electrophoresis with a newer batch of cDNA then we were able to use. His

research gave significantly different results that altered the way we continued on in experiments.

Dr. Smith’s gel showed the C1 band was at the correct size without the presence of primer dimers,

so this will be the sample that we continue on with.

After transforming the cloned APN2 genes into plasmids by use of the pENTR/D-TOPO vector

kits, we were able to electronically construct plasmid maps of what the target plasmid should look


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like containing the cloned DNA. Gene Construction Kit 3.0 was used to create a plasmid image

and to place in restriction enzymes that will be useful in determining whether the plasmid contains

the APN2 gene. The restriction enzyme digests that were used to determine if the target plasmids

contain the DNA were NheI/EcoRI and XhoI/AvrII. After purifying the plasmids and digesting the

plasmids with these restriction enzymes, agarose gel electrophoresis was utilized to determine if

the cloned genes were present in the samples. As seen in Figure 4, samples 1-5 in both digests

appear to have a distinct patter, but none have the correct band sizes that give lead to the

conclusion that the gene is present. In sample 6 or digest 2, the bands are correctly placed at

around 1,194 and 2,590 bases, which shows that the gene is likely in this sixth sample. Sample 6

was most obviously chosen to cryopreserve for future research because the cloned gene is likely in

the plasmid vector. Sample 5 was also chosen to cryopreserve so that further experimentation on

these plasmid vectors might shed light on what is in samples 1-5 since a distinctive pattern was

visible. Cryopreservation will allow the samples to remain viable for experimentation and research

for 10-15 years.

Conclusions

Figure 1, the genomic sequence of the Tetrahymena homolog APN2, shows the start and stop

codons, primers, introns, exons, and ESTs. Because of an underlined “G” that is predicted to be an

intron after the original start, another start was found in case the first was wrong. Later,

polymerase chain reaction showed that the original start was in fact right and that the underlined

“G” may have been a mistake when the sequence was produced on the Tetrahymena Genome

Database Wiki, http://www.ciliate.org/. Figure 2 shows two agarose gel electrophoresis captures

after polymerase chain reactions were performed. These pictures show that both the genomic DNA

and cDNA were correctly predicted at 1,478 bases and 1,191 bases respectively, so the cDNA was


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chosen to continue on in research because the cDNA does not contain the introns. Figure 3 shows

the plasmid map that was generated using the computer model Gene Construction Kit 3.0. The

restriction enzymes that were selected included digest 1 of Nhe1 and EcoRI and digest 2 of XhoI

and AvrII. Figure 4 is the agarose gel electrophoresis capture after restriction enzyme digestion.

Samples 1-5 do not appear to contain plasmids with the cloned gene, but do have a distinct pattern

worthy of further research, so sample 5 was chosen for cryopreservation. Sample 6 of digest 2

does appear to contain the cloned gene because the bands were predicted correctly at 1,194 and

2,590 bases. Sample 6 was also cryopreserved in glycerol.

Noticeable mistakes in this research seem to have remained minimal because the majority of any

mistakes that occurred were able to be fixed without it affecting the experiments in any way.

Errors that might have affected our research were usually small pipeting errors that may have

slightly affected the final amounts of a solution, but typically this has had no affect on the overall

outcome. Data and information from previous research on APN2 and its homologues in a variety

of species show that APN2 plays a major function in DNA repair, and because of role, APN2

might have a possible link to various cancers. If APN2 is correcting the DNA damage that occurs

to cancer cells via chemotherapy and radiation treatments, then inhibiting the work of APN2 may

increase the success rate of cancer treatments in medical patients and thus increases the chances

that cancer patients will go into remission. Further research into APN2 could be very beneficial in

cancer research and is an excellent candidate for additional experimentation. For future testing on

possible genes linked to cancer, the specific functions of a gene would be very useful in simply

understanding why this research could be essential or beneficial. While the bioinformatics data is a

great resource throughout the entire research period, a better grasp on what each piece of

information from Lab 3 means from the start would also be positive improvement. It is easy to go

through these lab protocols without entirely knowing why each step was taking place, and possibly


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focusing on each lab after it is completed in order to comprehend why those steps were taken in

the grand scheme of the research would be very valuable. Now that I am able to look back on the

research and put all the labs together into one comprehensive study rather than individual

procedures, I have better understanding of the project in general. I am also more interested in this

research now that I see why I did experimentation with APN2 specifically than I was at the

beginning of the class, because I have a much better understanding and appreciation of the

implications that APN2 has in cells and the human body.


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References

Al-Attar, A., Gossage, L., Fareed, K., Shehata, M., Mohammed, M., Zaitoun, A., Soomro, I., . . .

Madhusudan, S. (2010). Human apurinic/apyrimidinic endonuclease (APE1) is a prognostic

factor in ovarian, gastro-oesophageal and pancreatico-biliary cancers. British Journal of

Cancer, 102, 704-709.

Bapat, A., Fishel, M., & Kelley, M. (2009). Going ape as an approach to cancer therapeutics.

Antioxidants & Redox Signaling, 11(3), 651–667.

Jiang, Y., Zhou, S., Sandusky, G. E., Kelley, M., & Fishel, M. (2010). Reduced expression of

DNA repair and redox signaling protein. Cancer Invest, 28(9), 885–895.

Ma, W., Resnick, M., & Gordenin, D. (2008). Apn1 and apn2 endonucleases prevent

accumulation of repair-associated DNA breaks in budding yeast as revealed by direct

chromosomal analysis. Nucleic Acids Research, 36(6), 1836–1846.

Smith, JJ. (2011). BMS 110. Section 999, Introduction to the Biomedical Sciences. Laboratory

Protocols.


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Figures and Tables


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Tube 1 Tube 2

Concentration 1.8224 µg/µL 3.6424 µg/µL

A260 36.448 72.848

A280 16.330 32.135

A260/A280 2.23 2.27

Table 1: Quantification and purification of Tetrahymena DNA. The results of the quantification of Tetrahymena DNA by the NANODROP 2000 can be seen in the table. Tube 2 was used to continue on in research because of the higher concentration, but also because the A260/A280 is slightly higher showing a slightly purer DNA solution.

examination of the apn2 homolog in tetrahymena...

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