Lisa Ehrenreich

Dr. Wilma Saffran

Department of Chemistry and Biochemistry

Queens College- CUNY

Chem291, Spring 2015

Repair of Psoralen-Induced

Interstrand Crosslinks in

PSY3-Deficient Yeast Cells

Interstrand Crosslinks (ICLs) are covalent bonds

formed between the two strands of DNA

ICLs are cytotoxic to cells because they prevent

DNA replication and transcription

If left untreated, they can be fatal to the cell

Cells have different mechanisms to treat ICLs

Interstrand Crosslinks in DNA1

ICL Repair2

ICL can be repaired using three different pathways:

Nucleotide Excision Repair (NER)

Post Replication Repair (PRR)

Homologous Recombination (HR)

Post Replication Repair (PRR)

Allows the tolerance of a lesion rather than its removal and enables DNA replication to continue

Two pathways exist for PRR:

Error Prone PRR: trans-lesion synthesis, which allows the DNA machinery to replicate past the DNA lesion using a specific trans-lesion polymerase

Error Free PRR: a template switch process in which the undamaged sister chromatid is used as a template for the damaged sister chromatid

Homologous Recombination (HR)

The major repair pathway for ICL repair

Exchanges genetic information between

homologous DNA sequences to repair

damaged DNA molecule

Triplication

Gene deletion

Gene conversion

Conservative

Most preferred

Three Pathways of Homologous Recombination

Gene conversion and gene deletion

Triplication

Psoralen3

ICLs were induced by addition of psoralen

Psoralen intercalates into DNA and, upon exposure to UV radiation, it can form covalent ICLs with thymines

Although psoralen is a carcinogen, it is used in the treatment of psoriasis using small doses

Catalyst.berkeley.edu

PSY3 Gene4

PSY3 gene is involved in ICL repair

Psy3 protein interacts with other proteins (Shu1, Shu2,Csm2)

to make the Shu complex

Error-free PRR utilizes the Shu complex to facilitate HR in

order to accomplish template switching

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

Survival Experiment

Saccharomyces cerevisiae (bakers yeast) underwent psoralen addition followed by exposure to UV light to induce ICLs

Survival level can be determined by diluting and plating yeast cells on YPD plates and then counting the colonies

Survival curves can then be compared between repair deficient and repair proficient yeast

Survival level of the

PSY3-deficient strain

decreases as psoralen

concentration increases

PSY3-defiecient strain

is more sensitive to

psoralen induced ICLs

than the repair

proficient strain

PSY3-deficient strain is

about as sensitive to

psoralen induced ICLs

as RAD51-deficient

strain, and more

sensitive than RAD57-

deficient strain, which

have partial

recombination activity.

0.01

0.1

1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Re

lati

ve S

urv

ival

[Psoralen] (uM)

psy3 rad51 HR deficient rad57 HR deficient Repair Proficient

rad51 HR deficient

rad57 HR deficient

repair proficient

Psy3 HR deficient

Relative Survival

Recombination Assay

Measure Recombination

Yeast cells engineered to have functional TRP

gene flanked by nonfunctional his genes

Plate cells on His- plates (plates containing all

nutrients except amino acid histidine)

Colonies that survived had undergone

recombination to become His+

Functional HIS gene

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

10

00

[Psoralen] (uM)

psy3 rad51 HR Deficient rad57 HR Deficient Repair Proficient

PSY3 genes underwent more recombination than both the repair

proficient gene and the repair deficient RAD51 and RAD57 genes

His+ Recombination

psy3-deficient

rad51 HR deficient

rad57 HR deficient

repair proficient

Genetic Analysis

Determine what percent of the recombinant population

underwent deletion

Transfer the His+ colonies from His- plates onto Trp- plates

(plates containing all nutrients except amino acid

tryptophan)

Cells that had undergone gene conversion or triplication can

survive while cells that had undergone deletion cannot

survive

Compare deletion rates of repair-deficient and repair-

proficient cells

Functional TRP gene

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

ele

tio

ns

[Psoralen] (uM)

-Trp Deletions

Psy3 Repair Proficient

psy3-deficient

repair proficient

Genetic Analysis

Psy3-deficient cells and repair proficient cells exhibited about equal amounts of deletions.

Physical Analysis

Determine which of the 3 recombination pathways occurred more frequently

Extract and purify DNA from PSY3-deficient yeast cells

Amplify DNA via PCR

Gel electrophoresis to determine presence of DNA in our samples

Xba1 digestion

Gel electrophoresis to determine if DNA samples underwent triplication,

deletion, or gene conversion

Physical Analysis Deletion is

about the same

in repair

proficient and

repair deficient

cells

Gene

conversion is

main

recombination

pathway for

repair-proficient

cells but not for

repair-deficient

cells

Triplication is

increased in

repair-deficient

cells

PSY3-deficient

strains have

higher change

in gene copy

number

7%

25%

68%

8%

53%

39%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Deletion Gene Conversion Triplication

Recombination in PSY3 Repair-Deficient and

Repair-Proficient Yeast Cells(-) Psoralen

11%

47%42%

4%

84%

12%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Deletion Gene Conversion Triplication

Recombination in PSY3 Repair-Deficient and

Repair-Proficient Yeast Cells(+) Psoralen

Deletion Gene Conversion Triplication Deletion Gene Conversion Triplication

%R

eco

mb

inat

ion

%R

eco

mb

inat

ion

PSY3 Repair-Deficient

PSY3 Repair-Proficient

Conclusion

PSY3-deficient yeast cells have lower levels of survival, higher levels of

recombination, and more changes in gene copy number.

PSY3-, RAD51-, and RAD57-deficient yeast strains all had lower levels of

survival. However, unlike RAD51- and RAD57-deficient strains, the level of

recombination in PSY3-deficient strains is increased. This suggests that PSY3

deficiency leads to inappropriate recombination that does not contribute to ICL

repair and survival.

This is supported by higher levels of triplication in response to ICL induction.

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.

Practical applications

References1. 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. 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

5. Page, et al. The Yeast Shu Complex Utilizes Homologous Recombination Machinery for

Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue. 2013.