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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+
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
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.