Role of DNA repair enzymes
in removal of bulky DNA
adducts in zebrafish
Jennifer Atkin
Dr. William Baird
Significance
Smog!
Cigarette smoking!
DNA Adduct Formation

Polycyclic Aromatic Hydrocarbons (PAH) such as DBP
are environmental carcinogens found in cigarette
smoke, diesel exhaust, and other products of
combustion.
3
2
4
5
1
O
6
14
7
13
12
8
11
10
9
Cytochrome P450
and epoxide
hydrolase
D BP
Cytochrome P450
HO
OH
DBP-dihydrodiol
CH2OH
O
HO
N
N
N
N
NH
HO
HO
OH
Adenosine (blue) adduct formed by reaction of
Dibenzo[a,I]pyrene (red) with DNA
HO
OH
DBP-diolepoxide
DNA Repair
XPA
XPG
XPG
CSA
= DNA adduct
Goals of the Study



To identify and clone the Zebrafish DNA repair
enzyme genes, XPA, CSA, and XPG.
To determine the levels of expression of these
enzymes at different stages of development in
Zebrafish.
To evaluate the role of the repair enzymes in
effective removal of bulky DNA adducts caused
by the environmental carcinogen
Dibenzo[a,l]pyrene (DBP).
Identifying and cloning genes
Search for the genes on the web
(www.ensembl.org)
Design primers and amplify the genes via PCR
Sequencing
Zebrafish Repair Gene Sequences
XPG
GTAACTTTACTTGGTGGTAGAAGCGCGAGCAAAAGCTCTCGGGTGACAGGCTGAACTCTTTTAAACCATCTTCCTCAGACATCTGTCCTTCCAGACAGACAACATGGGTATTCAGGGCCTCTTGCAGTTTATCAAAGATGCTTCGGAG
CCCATGCATGTGAAGAAGTATCGAGGACAGACAGTGGCTGTGGACACATACTGCTGGCTTCATAAAGCAGCTTTTTCATGTGCAGAGAAGCTTGCAAAAGGGGAACCTACAGATCAGTATGTCTCCTACTGTATGAAGTTTGTGGACA
TGCTGCTTTCTTTTGGTGTTAAACCTATCTTGGTGTTTGATGGTCGTAACTTGCCCTCCAAACAGGAAGTGGAGAAGTCCCGGCGAGAGAGGCGACAGGCCAATCTGCAGAAAGGCAAACAGCTGCTGCGGGAGGGAAAGATAACAGA
AGCCAGAGAATGTTTTACCCGAAGTGTTAACATTACCCCATCTATGGCACATGATGTCATTAGGGCTGCCAGAACACGTGGAGTGGACTGTGTTGTTGCTCCATATGAAGCAGATGCACAGTTAGCTTTCCTTAACAAATCTGATATC
GCTCAGGCGGTGATCACAGAGGACTCAGATCTGCTGGCATTTGGCTGCAAAAAGGTCATTTTAAAGATGGATAAACAGGGTAATGGATTGGAGATTGAGCAGTGTCACCTGGGCCGATGCAAATCTCTTGGCAACATCTTCACAGAGG
AGAAGTTTCGATATATGTGCATCCTCTCTGGCTGTGACTACTTGCAGTCTCTGTATGGTATTGGTCTGGGAAAAGCATGCAAGCTGCTCAGGATGGCAAATAACCCTGATATCCTGAAGGTGATCAAGAAGATGGGGCAGTATTTGAA
GATGGACATTTCAGTTCCAGAAGAATACATTGAGGGTTTCACAAAAGCCAACAACACATTTTTGTATCAGCTGGTCTTTGACCCCTTGAGGAGAAAGGTGGTACCTTTGAACCCTTATCCAGATCACATTAATCCTGCTGCCCTCAGC
TATGCTGGAACTAACGTAGGTGATGAGAAAGGGCTGCAAATGGCTCTTGGGAACCTGGACATTAACACAATGCAAAGAATAGATGACTTTAACCCTGACGCTCCTCAAACTCAGCCACCTAAAGCTCCTCGCAGTAGTTCCTGGAATG
ACAGATGTGACAAAACTGCCACAACACAGGCAAGCATTTGGAGCCAGAACTATGAACCAGGTTGCACCAAGCCACAGTCTCCTACCTCCCCTAAAAGACCACCACCCACCCGGGGCAAAGAGAGGATTGTTAGTGTGCAGAGTCTAAA
GTTGCCCCAGAGAGAGTCACAGGTGAAAAGACCCCGTGAAGACACAAGTCTCTCTGTGGATGACCTGTTAGAACAGTACACGGCTGGTGTAAAGAGACACTGTCCTGAAACTCAGCCCACAACTAAGCCGCTGACTAATGATAACATG
GTTTCTAAGGAGAATCATTGTGGCAGCACATCTGGACCATCTCGTCCTCGAAATCGCTTTGCAACCCTCTTGCAGTGGAGAAACCGATCAGAAGAGGGCACTGAAGAGCAGGGCACATGCAGCAGGTTCTTCTGTCATGATGAATCCA
ACATAGCAGAGACACAGGAAGACTCAAAACAAGACTCTTCACAGTCTGTGGAGTCTCAAGAGAAGCATGTGTCACAGTCTGGAGGAGACACATCATCACTGTGTGAAGACCCAGAGAGAGAACAGGACAAAGATGAGCCATCTAGCCC
ACCCGCATCTCCATCTTGCTCTTCCAGGCCTGCTAGTGTGGGTCTTGGGGTCTTCAGCTGGTCAGGGAGCACCAAAGAACTCAATAAATCTGTTTCACATCCAGCCAGAGACAGCACAGAGAGACAGCGGTCATCCTCCACACCTTCA
GGATTATCTACACTGCAGCAGTTTCACAGAAATAAGGCAAGAATCTCCTGGGCAGGACCTGGGCTTTCTCTCTCGTCCTCACCTGTTGAAGGCAGTGAGGATGCTGGAAATTCTCCCGGGTCTCCTCCATCTCAGGACAGTGCCTATT
TCTCCCAGTCCAGCAGTATCTCTGCTAGTGTGGAGAACTCTTTAGTCACAGAAGACAACTCTGATAAGGAAAAGGAGAGAGATTCAGACGTCTCCAACAGCCCTAGCAGCTCTCCACTAGACAGGCTGAAACCTGCTGTTAACAGAAC
AAAGGCAAGGTTGTCCCGGAAAGGGGCTTGTGGGCAGGGTAAAGGCGGTAAAATTGAGACCTCCGCTCCTGCTAGAGCCAGCGGTCTGAGAAGGAAACCAAGCGGGAAGAAAAATGTCAACAATGAGAACAGCCCAGGTCTGCAGGCC
ACCATCAGTGGCCTCTGGGGGGCCTTCAGCTTCAAGAAGGACAGTCCAAAGTTAAGTGCAACCAAGAAAGGAGAGCCGATGTCACCGGTTGGGGAGAATGTGGTGATGGAAACGACTCAAGCTGATAAAGAGATTTTCATCATTGCTG
AATGACATGTCACTGTTTCAACTGAATAACTTTTTTAAGAGTATCAGACAGTCTTTCAGACATACTGTGTATGTTAATGATTTGTAAGATAAATCAAACTTTTGTAAAATTATGAATTTTATTTCAAGTCAATAAAGACGTTATTCTT
TA
XPA
ATTATTAACATTTTCATTTAAGTTCTCTTTTGACTTAACGCTGATCTGGTAAGACAAATCTTCATGAACACCTCGCTAGCTGATCATGGAGATGCCAGAGGCCAGTCATTGTCAACACACATCGCCAGACTCTGCAAACTCTTCAAAT
GAGTCCAGCATGTCTTTAACACCACACATGCTGGCCAAAATTGAGCGAAACCGCCAACGGGCGCTGATGCTCAGACAAGCCAGACTCGCCAGCAGACCGTCCTCCGCTGTAGAAGGAGCTACTCACGCTAAAGTGGCAAAAACTATAG
ACTCTGGTGCTGGGTTTTTTATTGAAGAGGAGACAACAGAGGACGAGCAGCAGGAGAAGAGAGTCGTTCAACAACCAGCCCCAGTGATGGAGCCTGATTATCTGATGTGCGAAGAGTGTCAAAAGCCATTTATGGATTCTTATCTGAG
CAACAGTTTTGATCTGTCTGTGTGTGATAAATGCAGAGACAATGAAGTAAAGCACAAGCTAATATCTCGCACGGAAGCGAAAAAGAATTACCTCCTGAAGGACTGTGATCTGGATAAGAGGGAGCCACCGCTGCGATTCATCCTGAGG
AAAAATCCTCACAATCCACGCTGGGGAGACATGAAACTCTACTTAAAGACACAGGTTGAGAAACGCTCTATGGAGGTCTGGGGCAGCGATGAGGCTCTGGAAGAGGCCAAGGAGACTAGAGAGGAGAACAAAGAGGTGCAGAAGCAGA
AACGCTTTAATAAAAAAGTCAAAGAGCTGAGGCGGACGGTGAGGAGCAGTATGTTTAAGAAAGACACCAGCATTCATCAGCACGACTATGGCCATGACGAGCTGCTGGATGAAGAGGAAGATTCTTTCAGTGAAAAGTGTAACTTCAG
AGATCCTACCTGTGTAAATACAGCCAACATTAGCACTCAGAAGATGTTTTCTAGTAGAAACTACACCTTAAAAACGCACAAATCCCCTTTATCCTTGAGAATATGTCAGAAAGGTGAAGAAAAGCAACAGAAAGATCTGCAGCAGCCA
GACTCTGACAATTATCTAACCATCCCCATTAAAGCTCATCCTTCAGATTCAAAACCCTCACATGCATCGGGCTGCACCAAGTCTCACCTAAATACCTCCCACCTTCAGCCCGTGCAGCGCTCGCCTGTCATAATGGAGTCTTGGTCCC
CTGACAGCCCTACAGCTATGATTTATCATCACGCTGTGCCCGTGCCAAATGTACCTGTTGTTCAACCCCAGCTGCTCTGCCTCACGCCCCCTGCAGACGTCCCGCATGCTCACATCCAGCGCAAGCTGCTGCTTGACCCCACCACGGG
TCAGTACTACTTAGTGGACACCCCTGTTCCGATGCAGCCCGCCGCCCAGAGGTTTTACCATCCTGAGAGTGGCCAGTACTTGGACATTCCTTTATCACCACTCCAGTGCCAGTGTCGGTGTCACCGGTCGCCCTCGGTCCTGCAGCCT
ATCCCCCAACTTACTTGGTCTACCCTTCGACCTTTCTGCCTGCACCCCCGGCGCCGCAGTCACATTCATCCACCTGCTCCGAGGGCGAGGATGCAGTGGAGACGGGCAGCATGTATATGATCCCGCCGGGCAGCACAGCACCCAGCAG
CACCACCAAACCGGTCATCAGCATCACATCGCAGCAAGGTGCCCGTATCGTTGCTCCTCCCTCCTTTGATGGCACAACAATGAGCTTTGTGGTGGAGCATCGATAACGGAAGGAGGATCCCAGTCAGAGTGTGTTCACCAACCACTTC
ATCAAAAAAAAAAAAAAAAA
CSA
AAACACAAGTCCAATGTCTCTAAAACAATGTTAAGTTTTCTTTACGCAAGGCAAACTGGGCTTGACGACCCAGTTCGACTGAGACGTGCAGAATCCACACGCAGGGTGCTGAGTCTTGAGTTGAACCACAACAGGGATGTGGATCGTA
TTCATGGCAATGGAATCAACACCCTTGACATTGAGGTTATTGATGGAAGATATATGCTGTCAGGCGGATCTGATGGCGTAATTGTGATTTATGACTTAGAGAACAACAGCAAGAAGCCTCAATACACCTGCAAAGCAATCTGCACTGT
GGGCAGTCGACATGTGCATAAATTCAGCGTGGAAACAGTGCAGTGGTACCCACATGACACCGGTATGTTTGTATCCAGCTCTTTCGACAAAACCATGAAAGTCTGGGATGCTGAAACTCTGAAGCCAGCTGATGAGTTCCAGTTTGAT
GGCAATGTCTACTGTCATCACATGTCTCCTATAGCCCGGAAGCACAGCCTTGTGGCAGTTGGTACCAAAGACCCCAAAGTGCAGCTTTGTGATCTGAAGTCTGGGTCGCGTATCCACATCCTTCAGGGTCACAGGGGCGAGATCCTCT
CAGTGAAATGGTCTCCACGATATGAACACATACTGGCCACTGCCAGCACTGACAGTAGGGTTCGCATCTGGGATGTGAGAAGAGCCTCCGGGAGTCTTTTTACTTTGGACCAGCACAATGGAGATAAATCCAAGGCCTCGTCTGAAGC
AGTGAACACAGCACACAACGGAAGAGTGAATGGGTTGTGTTTCACAGCTGATGGACTCCACCTCCTCACTACAGGGACAGATGATCGCATGCGTCTGTGGAATAGCGGCACTGGGGAAAACACATTAGTGAACTATGGGAAGGTCGTG
AACGAGAGCCGCAAAGGTCTGAAGTTTACCGTATCAAGGGGTTGCAGTCCAGAGTTTGTGTTTGTGCCATGTGGAAGCTCAGTAGCGGTGTATGGGCTTCATTCTGGAGAGCTGATCACTATGTTAAGGGGACATTATAATAATGTGG
ACTGCTGCGAGTTCCACCCTGATTATCAGGAGCTGTACAGTGGAGGTAAAGACTGTAACATACTTGCCTGGGTTCCAGTGTTAAGACAGCCTGATGTTGATGATGAAGAAACCAACACGAAACGGGGTGGAACGCAAGCTGCTATCAA
TCCTGCTTTTGCCGATGCCTGGAGCAGCGACGAAGACTGACAGATCAACCCAGCAATGTTTTTTTATATCCAAATACACCACTTTATACATTATTAAAGAAGTCAAAGTGCTCTTATTGCATTCAGAGGCACAGCAGGTGCATATAAG
CTCAATGTACAGAAAAAAAAGACTTGTGGGAGATTTGTTCTCATTGCTTGTGACCATCTGATTTGCATG
Repair Enzyme Homology between
different species
Fugu
X. laevis and X. tropicalis
CSA: 92%
CSA: 86%
XPA: 86%
XPA: 64%, 78%
XPG: 92%
R. norvegicus
XPG: 60%
M. musculus
H. sapiens
CSA: 86%
CSA: 84%
XPA: 72%
CSA: 85%
XPA: 73%
XPG: 61%
XPA: 75%
XPG: 48%
XPG: 60%
XPA Phylogenetic Analysis
XPA Phylogenetic Analysis
CSA Phylogenetic Analysis
CSA Phylogenetic Analysis
Real Time PCR
RT-PCR and amplification of XPA, CSA and XPG cDNA
Total Zebrafish RNA
Oligo dT primers
Superscript RT
RNase inhibitor
primer
primer
SPP
First strand cDNA
Amplify cDNA
SPP
PCR
Amplified product
SPP: Specific pair primer for each gene
CSA
XPA
XPG
12 3 4 5 6 12 3 4 5 6 12 3 4 5 6
water
Real Time PCR
1: STD control
2: 12 hr
200 bp
3: 24 hr
4: 36 hr
5: 48 hr
6: (-)RT control
Experimental Scheme
Treat with
DBP
Microinjection
embryo
Protein
Western Blot
embryo
Treat with
DBP
DNA
Protein
DNA
DNA
adduct
analysis
Western Blot
DNA
adduct
analysis
Translation inhibition
Morpholinos
tRNA
Protein
60S
~~ G A C A A C A U G G G U A U U C A G G G C C U C U U G C A G U U U A U C A ~~
40S
60S
Protein
40S
G T T G T A C C C A T A A G T C C C G G A G A A
~~ G A C A A C A U G G G U A U U C A G G G C C U C U U G C A G U U U A U C A ~~
Morpholinos

Inject the designed morpholino into the developing
embryo at the 1-cell stage.

Once incorporated into the embryo, the morpholino
blocks translation of the gene.
O
HO
O
OH
O
O
HN
O
Morpholino
A
O
O
48 hpf
3’-Carboxyfluorescein
Future Work


Continue with morpholino injections and
establish that the genes are knocked down.
Isolate DNA from injected embryos and look
for any change in amount of DNA damage
caused by DBP.
Acknowledgements
Howard Hughes Medical Institute
Kevin Ahern
William Baird
Robert Tanguay
Brinda Mahadevan
Eric Andreasen
Mark Reimers
Jane LaDu
Lijoy Mathew
Eric Brooks