Thesis Paper

Repair of Psoralen-Induced Interstrand Crosslinks in Psy3-Deficient Yeast Cells

Lisa Ehrenreich

Department of Chemistry and Biochemistry

Dr. Wilma Saffran

Queens College- CUNY

Chem291, Spring 2015

REPAIR OF PSORALEN-INDUCED INTERSTRAND CROSSLINKS

IN PSY3-DEFICIENT YEAST CELLS

ABSTRACT

The current study focused on the survival and recombinants of psy3-deficient yeast cells in

response to psoralen-induced interstrand crosslinks (ICLs). ICLs are cytotoxic to cells.

Homologous recombination (HR) is the main repair pathway of ICLs in cells. The three

pathways of HR are gene conversion, deletion, and triplication. Psy3-deficient yeast cells

decreased in level of survival with increasing psoralen concentration. Psy3-deficient yeast cells

exhibited lower levels of survival and higher levels of recombination compared to psy3-

proficient yeast cells as well as compared to rad51- and rad57- deficient yeast cells. This

increase in recombination in conjunction with decreased level of survival suggests that the

recombination was not conducive to ICL repair or cell survival. There were similar levels of

deletion in psy3-deficient strains compared to psy3-proficient strains. The main mechanism of

repair in psy3-proficient strains was gene conversion. Conversely, there were increased levels of

triplication in psy3-deficient strains. This increase in triplication supports the suggestion that the

increased level of recombination was not conducive to cell survival because triplication causes

an increase in gene copy number and does not maintain DNA integrity.



Repair of Psoralen-Induced Interstrand Crosslinks in Psy3-Deficient Yeast Cells

Cellular DNA is susceptible to chemical modifications. These modifications can be

caused by environmental agents or by endogenous agents produced by cellular processes. Many

of these changes, in the form of phosphate or base changes, occur on either one or the other

strand of DNA. Psoralen, however, is an agent that forms covalent bonds with both strands of

DNA, forming what is called an interstrand crosslink (ICL). ICLs are cytotoxic to cells because

they prevent DNA replication and/or transcription. If left untreated, ICLs can lead to cell death.

It has been estimated that as few as 20 ICLs in the bacterial or mammalian genome can be lethal

to cells that lack the ability to remove the crosslinks.1

ICLs can be repaired using three different pathways: nucleotide excision repair (NER),

post replication repair (PRR), and homologous recombination (HR). The NER pathway involves

the recognition and removal of a damaged DNA by exonucleases, resulting in a single strand gap

that is filled in by DNA polymerase using the undamaged strand as a template. PRR occurs after

the DNA has already been replicated. This pathway allows the tolerance of a lesion rather than

its removal. Two pathways exist for PRR. Error prone PRR accomplishes trans-lesion synthesis,

allowing the DNA machinery to replicate past the DNA lesion using a specific trans-lesion

polymerase. Error Free PRR entails a template switch process in which the undamaged sister

chromatid is used as a template for the damaged sister chromatid. Homologous recombination

entails exchanging genetic information between homologous DNA sequences to repair the

damaged DNA molecule. These three mechanisms of ICL repair can be coupled in order to

maximize results.2

Homologous recombination is the major repair pathway for ICL repair. There are three

possible pathways for HR: triplication, deletion, and gene conversion. Triplication occurs when

homologous chromosomes do not align properly, and there is unequal sister chromatid exchange.

This pathway results in an increase in gene copy number. Deletion occurs when a gene is cut out

and flanking genes are annealed. This pathway results in a decrease in gene copy number. Gene

conversion occurs when there is a mutation in a gene. This pathway is conservative, meaning it

results in an unchanged gene copy number. Gene conversion is the most preferred method

because it maintains genome integrity by not causing a change in gene copy number. Figure 1

shows the three possible pathways of HR.3



Figure 1. Triplication, gene conversion, and deletion.

Psoralen, a natural plant derivative, was used in the current study to induce ICLs in yeast

cells. Psoralen works by inserting itself in between two strands of DNA. Without light, psoralen

does not interact covalently. However, upon exposure to UV light of 350nm, a covalent bond

forms on either side of the psoralen molecule with thymines on either strand of DNA. This

creates an ICL which is cytotoxic to the cell. While psoralen is a carcinogen, it is currently being

used in small doses to treat patients with psoriasis by inducing ICLs in skin cells in order to stop

the excessive skin cell replication that causes the red or white patches of skin on people with

psoriasis.4

The gene of focus in the current study was the psy3 gene. This gene is involved in ICL

repair. The psy3 gene codes for the psy3 protein, which interacts with other proteins, shu1, shu2,

and csm2, to form the Shu complex.5 Error-free PRR utilizes the Shu complex to accomplish HR

in order to facilitate the template switching involved in error-free PRR. The Shu complex thus

couples HR to error-free PRR. Deficiency in any of the genes comprising the Shu complex leads

to deficiencies in damage repair.6

In order to study the effect of a deficiency in the psy3 gene on survival of the cell, a

survival experiment was done on a psy3-deficient yeast cell. The psy3-deficient yeast cell was

treated with psoralen and exposed to UV light. Survival level was then determined by diluting

and plating yeast cells on YPD plates and then counting the number of colonies that grew.

Survival curves were then compared between repair deficient and repair proficient yeast cells.

The results of the survival experiment can be seen in Figure 1 below.

Concurrently, a recombination assay was conducted. This was to see how much

recombination occurred. The yeast cells were engineered to have a functional trp+ gene

surrounded by nonfunctional his- genes. These cells were plated on his- plates. The YPD plates



that were used to test survival contained all the essential nutrients and amino acids needed for

yeast survival. The his- plates, however, contained all the nutrients necessary for yeast cell

survival except for the amino acid histidine. Only cells that contained functional his+ genes could

produce the histidine amino acid on their own and be able to survive on the his- plate. If any type

of homologous recombination had occurred, whether it was triplication, deletion, or gene

conversion, it would result in the conversion of the nonfunctional his- gene to a functional his+

gene, and thus allow the cell to survive on the his- plate. By studying the survival levels of yeast

cells on his- plates, we were able to compare the levels of recombination in repair deficient and

repair proficient yeast cells. The results of the recombination assay can be seen in Figure 2.

A genetic analysis test was then run. The purpose of this test was to determine what

percent of the recombinant population obtained in the recombinant assay had specifically

undergone deletion. The his+ cells obtained in the recombination assay were transferred from the

his- plates to trp- plates. These plates contained all of the nutrients necessary for yeast cell

survival except for the amino acid tryptophan. Only the cells that had undergone deletion could

not survive on these plates because out of the triplication, deletion, and gene conversion

recombinants, deletion recombinants were the only cells that had lost the trp gene and thus could

not synthesize the tryptophan amino acid necessary for survival that was lacking from the

medium. By studying the survival rates of his+ yeast cells on trp- plates, we were able to compare

deletion rates of repair-deficient and repair-proficient cells. The results of the genetic analysis

can be seen in Figure 3.

Lastly, a physical analysis test was done in order to determine which type of homologous

recombination pathway- either triplication, gene conversion, or deletion- had occurred most

frequently in each strain of yeast cells. This was done by first extracting and purifying DNA

from psy3-deficient yeast cells. The DNA was then amplified via PCR. A gel electrophoresis was

run in order to confirm the presence of DNA in our samples. Bands on the gel indicate that there

is DNA present. After this, Xba1 digestion was performed. Xba1 is a restriction enzyme that

recognizes and cuts certain sequences in DNA. Finally, a gel electrophoresis was performed in

order to determine if the DNA samples had undergone triplication, deletion, or gene conversion.

If triplication had occurred, there were five bands on the gel; if gene conversion had occurred,

there were three bands on the gel; and if deletion had occurred, there was only one band on the

gel. Results of the physical analysis can be seen in Figure 4.



Methods

I. Survival

1) Plate preparation

a) YPD Plates: In a 2L flask, add 1000mL of distilled water followed by addition of 10g

of yeast extract, 20g of peptone, 20g of dextrose, and 17g of agar. Then, cover the flask with

aluminum foil and autoclave under media for one hour. Once the autoclave is finished, shake the

flask vigorously to ensure the agar has dissolved. Next, let it cool to room temperature and pour

into plates. The plates are color coded black and red to identify them as YPD plates.

b) His- Plates: In a 2L flask, add 800mL of distilled water followed by addition of 6.7g of

yeast nitrogen base (YNB), 20g of glucose, 16.7mL of adenine, 16.7mL of luecine, 8.3mL of

tryptophan, and 8.3 mL of uracil. Then, add 200mL of distilled water and also 20g of agar. Then,

cover the flask with aluminum foil and autoclave under media for one hour. Once the autoclave is

finished, shake the flask vigorously to ensure the agar has dissolved. Next, let it cool to room

temperature and pour into plates. The plates are color coded green, black and orange to identify

them as His- plates.

c) Trp- Plates: In a 2L flask, add 800mL of distilled water followed by addition of 6.7g of

yeast nitrogen base (YNB), 20g of glucose, 16.7mL of adenine, 16.7mL of luecine, 8.3mL of

histidine, and 8.3 mL of uracil. Then, add 200mL of distilled water and also 20g of agar. Then,

cover the flask with aluminum foil and autoclave under media for one hour. Once the autoclave is

finished, shake the flask vigorously to ensure the agar has dissolved. Next, let it cool to room

temperature and pour into plates. The plates are color coded green, black and purple to identify

them as His- plates.

2) Cell Growth and Preparation: Colonies with deficient strain were transferred to sterile test

tubes, which contain 2mL of YPD broth. The colonies were then incubated at 30oC while shaking

overnight. The test tubes were then centrifuged at 2000rpm for five minutes to extract yeast cells.

The supernatant was decanted. Next, added 2 mL of sterile water into culture tubes and added

yeast cells. Vortexed to resuspend the yeast cells. The yeast cells were then stored on ice until

treatment with psoralen.

3) Psoralen Treatment: The yeast cells are treated with four different amounts of psoralen: 0, 5,

10, and 15 uL. The experiment was done in triplicates as A, B and C, so 12 microcentrifuge tubes

were used. The tubes were labeled as 0A, 5A, 10A, 15A, and the same was done for B and C.



Next, added 0.9mL of sterile water and 0.1mL of yeast cells into each labeled tube. Appropriate

amount of Psoralen, concentration of 0.023mM, was then pipetted into each corresponding tube

and then vortexed. Each set was then wrapped separately in aluminum foil and placed on ice for

30 minutes to allow the psoralen to enter cells and bind to DNA. The concentration of psoralen

that was added to the yeast can be calculated using the following formula:

4) Tube set up for Serial Dilution: During the 30 minutes waiting period, microcentrifuge tubes

were prepared for serial dilution. The 0 uL set would be diluted four times, so, the tubes were

labeled 0A 10-1 0A 10-2 0A 10-3 0A 10-4 and same for B and C. The 5uL set would be diluted

three times, so were labeled 5A 10-1 5A 10-2 5A 10-3 and same for B and C. The 10 and 15uL set

would be diluted only twice were labeled accordingly. Lastly, 0.9mL of sterile water was added

into each tube.

5) Irradiation: To prepare for the irradiation, 1 tube and 1 petri dish was labeled for each of the

12 tubes. In the cold room, a tray was filled with ice and placed under the UV lamp while the

overhead light was turned off and the yellow light was left on. The irradiation was done for each

set at a time. Each set was poured into its appropriate petri dish and placed under the UV lamp on

ice. A time was set for 10 minutes once the lamp was turned on. After elapsed time, the lamp was

turned off and the samples were pipetted into their corresponding labeled empty tubes.

6) Serial Dilution: Each of the 12 samples was then serially diluted as follow: 0.1mL from the

sample were pipetted into the 10-1 tube, then vortexed. Next, pipetted 0.1mL from the 10-1 tube

into the 10-2 tube, then vortexed. This was repeated for each dilution tube of all 12 samples.

7) Plating: The YPD plates were labeled the same as the serial dilution tubes and dated. For the

0uL psoralen samples, two sets of 10-3 and 10-4 dilutions were plated. All the others dilution tubes

were plated only once. Pipetted 0.1mL from each diluted onto the surface of corresponding plates

and were spread using sterile technique. The SD-his plates were labeled for each of the 12

undiluted tubes. Pipetted 0.1mL of undiluted tubes onto SD-his plates and spread out the cells.

Lastly, all the plates were incubated for two to three days until the colonies became visible.

8) Preparation for colony transfer: Yeast colonies were transferred from SD-his plates to YPD

plates using a 50-grid template. Two YPD plates for each SD-his plate was made, so a total of 100

colonies were counted but not all contained 100. Each plate was labeled accordingly: 0A-1, 0A-2,

0B-1, 0B-2, 0C-1, 0C-3, and same for the rest. The YPD plates, containing the recombinant cells,

were incubated for two to three days until the colonies became visible.



9) Transfer to SD-Trp Plates: Once the colonies were large enough, they could now be

transferred to SD-Trp plates. First, all the plates were labeled the same way as the YPD plates. The

YPD colonies were pressed on sterilized red felt and then the SD-Trp plates were pressed on the

felt, completing the transfer process. Did this for all the plates. The SD-Trp plates were incubated

for two to three days until the colonies became visible.

II. Physical Analysis

1) Yeast DNA Preparation for PCR: Yeast cells were inoculated for one to two days in 2 mL

of YPD broth while being shaken at 30o C. The tubes were then vortexed and transferred into 1mL

microcentrifuge tubes. After the tubes were centrifuged for two minutes, the supernatant was

decanted. The remaining pellet was resuspended in 500uL of 1M sorbitol and 0.1M EDTA.

Followed by an addition of 10uL of 2.5ug/mL of zymolase, each tube was then mixed by inversion,

and incubated at 37oC for 60 minutes. The tubes were then centrifuged for one minute. The tubes

were decanted, and the pellet was resuspended with 500uL of 50mM Tris and 20mM EDTA at

pH7.5 by vortexing. 50 µL of 10% SDS was then added to each tube and mixed by inversion. The

tubes were then incubated for the second time but at 65oC for 30 minutes. Next, added 200uL of

5M potassium acetate and mixed by inversion. The tubes were left either on ice for 60 minutes or

were refrigerated overnight.

Once cooled, the tubes were centrifuged for one minute and 600uL of the supernatant was

transferred into fresh corresponding microcentrifuge tubes and then added 600uL of isopropanol.

The tubes were left at room temperature for five minutes. The tubes were then spun for ten seconds,

the supernatant was discarded, and were left to dry upside down on a paper towel for five minutes.

Then, 300uL of TE and 30uL of 3.0M sodium acetate was added to each tube and mixed. 200uL

of isoproponal was added and mixed. The tubes were once again kept at room temperature for five

minutes, spun for ten seconds, and left to dry upside down on a paper towel. Finally, the 50uL of

TE was added to each tube and stored in refrigerator until PCR.

2) Polymerase Chain Reaction (PCR): A master mix was prepared, each enough for 25 PCR

samples, in a 1mL microcentrifuge tube. To each mix added:

630µL PCR water

240µL 5x Buffer

25 µL of each 10mM dNTP (dATP, dCTP, dGTP,dTTP)



180µL 25mM MgCl2

60uL of 5mM his3-1

60uL of 5mM his3-2

6 µL Taq Polymerase

Added 48uL of Master mix and 2uL of DNA template into PCR tubes. Ran the thermal cycler on

Program 2.

3) Preparation of 1% Agarose Gel: Into a 250mL erlenmeyer flask, added 100uL of 5X TBE

buffer, which was prepared by 900 uL of distilled water and 100 50X TBE, and 1g of agarose.

Swirled to mix and microwaved for two minutes in one minute intervals to guarantee a clear

solution. Wait to cool till near room temperature and poured into electrophoresis apparatus. Place

combs into apparatus to form wells and let solidify.

4) Xba1 Digestion: A restriction enzyme called Xba1 was used to perform Xba1 digestion. This

enzyme recognized and cuts the ...T↓C T A G A... site.4 A Xba1 mixture is made in 0.5mL

microcentrifuge tubes which includes:

XbaI Mix (enough for 18 samples)

330µL sterile water

60µL 10X Buffer

6µL 100X BSA

4µL XbaI

Add 20µL of XbaI mix into microcentrifuge tubes, 10µL PCR product. Incubate for one hour at

37oC. Run electrophoresis.



Results

Survival Test

Figure 1. Relative survival of repair proficient and psy3-, rad51-, and rad57- deficient yeast

cells with increasing psoralen concentration.

Recombination Assay

Figure 2. Levels of recombination in repair proficient and psy3-, rad51-, and rad57-deficient

yeast cells with increasing psoralen concentration.

0.01

0.1

1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Rel

ativ

e S

urv

ival

[Psoralen] (uM)

psy3 rad51 HR deficient rad57 HR deficient Repair Proficient

rad51-deficient

rad57-deficient

repair proficient

0

1

2

3

4

5

6

7

8

9

10

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

His

+ P

er 1

00

0

[Psoralen] (uM)psy3 rad51 HR Deficient rad57 HR Deficient Repair Proficient

psy3-deficient

psy3-deficient

rad51-deficient

rad57-deficient

repair-proficient



Genetic Analysis

Figure 3. Levels of deletion in psy3-deficient and psy3-proficient yeast cells.

Physical Analysis

Figure 4. Percentage of HR of each pathway in psy3 repair-deficient and psy3 repair-proficient

yeast cells without psoralen treatment (left chart) and with psoralen treatment (right chart).

7%

25%

68%

8%

53%

39%

0%

20%

40%

60%

80%

100%

Deletion GeneConversion

Triplication

Recombination in Psy3Repair-Deficient and

Repair-Proficient Yeast Cells(-) Psoralen

0

2

4

6

8

10

12

14

16

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5

% D

elet

ion

s

[Psoralen] (uM)

-Trp Deletions

Psy3 Repair Proficient

% Recombination

% Recombination

Psy3 Repair-Deficient Psy3 Repair-Proficient

11%

47%42%

4%

84%

12%

0%

20%

40%

60%

80%

100%

Deletion GeneConversion

Triplication

Recombination in Psy3Repair-Deficient and Repair-

Proficient Yeast Cells(+) Psoralen

psy3-deficient

repair-proficient



Discussion

Figure 1 shows the results of the survival experiment, which was performed in order to

compare the levels of survival in repair deficient and repair proficient yeast cells with increasing

psoralen concentration. Results showed that survival level of the psy3-deficient strain decreased

as psoralen concentration increased. This means that the psy3-deficient strain was sensitive to

psoralen. The psy3-defiecient strain was more sensitive to psoralen induced ICLs than the repair

proficient strain. The psy3-deficient strain was about as sensitive to psoralen induced ICLs as

rad51-deficient strain, and more sensitive than rad57-deficient strain, which have partial

recombination activity.

Figure 2 shows the results of the recombination assay, which was performed in order to

compare the levels of recombination in repair deficient and repair proficient yeast cells. As can

be seen from the results, psy3-deficient yeast cells underwent more recombination than both the

repair proficient yeast cells and the repair deficient rad51 and rad57 yeast cells. It is interesting

to note that according to the survival experiment above, psy3-, rad51-, and rad57-deficient

strains were all sensitive to psoralen and to ICLs, exhibiting decreased levels of survival with

increased psoralen concentration. However, unlike rad51- and rad57-deficient strains, the level

of recombination was greatly increased in psy3-deficient strains. Because psy3-deficient yeast

cells had a low survival rate but a high rate of recombination, this suggests that psy3 deficiency

leads to inappropriate, excessive recombination that does not contribute to ICL repair or survival.

Figure 3 shows the results of genetic analysis. Psy3-deficient cells and repair proficient

cells exhibited about equal amounts of deletions.

Figure 4 displays the results of physical analysis. Deletion levels were about the same in

repair proficient and repair deficient cells. This is in agreement with the results of the genetic

analysis test. Gene conversion was the main recombination pathway for repair-proficient cells

but not for repair-deficient cells. Triplication was increased in repair-deficient cells. Psy3-

deficient strains therefore incurred a higher change in gene copy number. This increase in

triplication supports the earlier suggestion that psy3-defiecient yeast cells undergo inappropriate,

excessive recombination that does not contribute to ICL repair and survival, because triplication

shifts all the DNA and is not conducive to survival.



Conclusion

In conclusion, psy3-deficient yeast cells had lower levels of survival, higher levels of

recombination, and more changes in gene copy number. Psy3-, rad51-, and rad57-deficient yeast

all had lower levels of survival. However, unlike rad51- and rad57-deficient strains, the level of

recombination in psy3-defiicient strains was increased. This suggests that psy3 deficiency leads

to inappropriate recombination that does not contribute to ICL repair and survival. This was

supported by higher levels of triplication in response to ICL induction, which causes an increase

in gene copy number and does not maintain DNA integrity. We can conclude that psy3 function

is important for the maintenance of genome integrity in response to interstrand crosslinking,

allowing recombination in a normal amount and in a pathway that is conducive to cell survival.

The cytotoxic effect of interstrand crosslinks forms the mechanistic basis of many

anticancer drugs in use today. ICLs are induced in cancerous cells to stop them from replicating.

Some cells, however, become resistant to the ICLs, so it is important to see how this resistance is

produced and specifically which genes are involved. With this knowledge, genes involved in ICL

repair can potentially be targeted so that ICLs can be induced to effectively stop cancerous cells

from replicating.



References

1. Lehoczky, P., McHugh P. J., Chovanec M. (2006). DNA Interstrand Cross-link Repair

in Saccharomyces Cerevisiae. Federation of European Microbiological Societies,

1-25.

2. Saffran, W., Ahmed, S., & Bellevue, S. DNA Repair in Defects Channel Interstrand DNA

Cross-links into Alternate Recombinational and Error-prone Repair Pathways. The Journal

of Biological Chemistry, 279(35).

3. Zheng, H., Wang, X., Warren, A. J., Legerski, R. J., Glazer, P. M., Li, Lei. (2006) Repair

of DNA Interstrand Cross-links: Interactions Between Homology-Dependent and

Homology-independent Pathways. DNA Repair 5, 566-574.

4. Noll, D., McGregor, T., Miller, P. (2008) Formation and Repair of Interstrand Cross-Links in

DNA. 277-301.

5. Ball, G. L., Zhang, K., Cobb, J. A., Boone, C., Xiao, W. (2009) The Yeast Shu Complex

Couples Error-Free Post-Replication Repair to Homologous Recombination

Molecular Microbiology 73, 89-102.

6. Page, et al. (2013) The Yeast Shu Complex Utilizes Homologous Recombination Machinery for

Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue.

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