Role of DNA repair Role of DNA repair enzymes in removal enzymes in removal

of bulky DNA adducts of bulky DNA adducts in zebrafishin zebrafish

Jennifer AtkinJennifer AtkinDr. William BairdDr. William Baird

SignificanceSignificance

Cigarette smoking!

Smog!

DNA Adduct FormationDNA Adduct Formation Polycyclic Aromatic Hydrocarbons (PAH) Polycyclic Aromatic Hydrocarbons (PAH)

such as DBP are environmental carcinogens such as DBP are environmental carcinogens found in cigarette smoke, diesel exhaust, and found in cigarette smoke, diesel exhaust, and other products of combustion.other products of combustion.

D B P

H O O H

H O O H

O

8 1 0 9 1 1

1 2 1 3

1 4 7 6

5 4

3 2 1

Cytochrome P450 and epoxide hydrolase

Cytochrome P450

DBP-dihydrodiol DBP-diolepoxide

NH

N

N

N

N

O

HO

HO

OHHO

Adenosine (blue) adduct formed by reaction of

Dibenzo[a,I]pyrene (red) with DNA

CH2OH

DNA RepairDNA Repair

XPA

XPG

= DNA adduct

CSA

XPG

Goals of the StudyGoals of the Study To identify and clone the Zebrafish DNA To identify and clone the Zebrafish DNA

repair enzyme genes, repair enzyme genes, XPAXPA, , CSACSA, and , and XPGXPG. . To determine the levels of expression of To determine the levels of expression of

these enzymes at different stages of these enzymes at different stages of development in Zebrafish.development in Zebrafish.

To evaluate the role of the repair enzymes To evaluate the role of the repair enzymes in effective removal of bulky DNA adducts in effective removal of bulky DNA adducts caused by the environmental carcinogen caused by the environmental carcinogen Dibenzo[Dibenzo[a,la,l]pyrene (DBP). ]pyrene (DBP).

Identifying and cloning Identifying and cloning genesgenes

Design primers and amplify the genes via PCR

Search for the genes on the web (www.ensembl.org)

Sequencing

Zebrafish Repair Gene Zebrafish Repair Gene SequencesSequences

GTAACTTTACTTGGTGGTAGAAGCGCGAGCAAAAGCTCTCGGGTGACAGGCTGAACTCTTTTAAACCATCTTCCTCAGACATCTGTCCTTCCAGACAGACAACATGGGTATTCAGGGCCTCTTGCAGTTTATCAAAGATGCTTCGGAGCCCATGCATGTGAAGAAGTATCGAGGACAGACAGTGGCTGTGGACACATACTGCTGGCTTCATAAAGCAGCTTTTTCATGTGCAGAGAAGCTTGCAAAAGGGGAACCTACAGATCAGTATGTCTCCTACTGTATGAAGTTTGTGGACATGCTGCTTTCTTTTGGTGTTAAACCTATCTTGGTGTTTGATGGTCGTAACTTGCCCTCCAAACAGGAAGTGGAGAAGTCCCGGCGAGAGAGGCGACAGGCCAATCTGCAGAAAGGCAAACAGCTGCTGCGGGAGGGAAAGATAACAGAAGCCAGAGAATGTTTTACCCGAAGTGTTAACATTACCCCATCTATGGCACATGATGTCATTAGGGCTGCCAGAACACGTGGAGTGGACTGTGTTGTTGCTCCATATGAAGCAGATGCACAGTTAGCTTTCCTTAACAAATCTGATATCGCTCAGGCGGTGATCACAGAGGACTCAGATCTGCTGGCATTTGGCTGCAAAAAGGTCATTTTAAAGATGGATAAACAGGGTAATGGATTGGAGATTGAGCAGTGTCACCTGGGCCGATGCAAATCTCTTGGCAACATCTTCACAGAGGAGAAGTTTCGATATATGTGCATCCTCTCTGGCTGTGACTACTTGCAGTCTCTGTATGGTATTGGTCTGGGAAAAGCATGCAAGCTGCTCAGGATGGCAAATAACCCTGATATCCTGAAGGTGATCAAGAAGATGGGGCAGTATTTGAAGATGGACATTTCAGTTCCAGAAGAATACATTGAGGGTTTCACAAAAGCCAACAACACATTTTTGTATCAGCTGGTCTTTGACCCCTTGAGGAGAAAGGTGGTACCTTTGAACCCTTATCCAGATCACATTAATCCTGCTGCCCTCAGCTATGCTGGAACTAACGTAGGTGATGAGAAAGGGCTGCAAATGGCTCTTGGGAACCTGGACATTAACACAATGCAAAGAATAGATGACTTTAACCCTGACGCTCCTCAAACTCAGCCACCTAAAGCTCCTCGCAGTAGTTCCTGGAATGACAGATGTGACAAAACTGCCACAACACAGGCAAGCATTTGGAGCCAGAACTATGAACCAGGTTGCACCAAGCCACAGTCTCCTACCTCCCCTAAAAGACCACCACCCACCCGGGGCAAAGAGAGGATTGTTAGTGTGCAGAGTCTAAAGTTGCCCCAGAGAGAGTCACAGGTGAAAAGACCCCGTGAAGACACAAGTCTCTCTGTGGATGACCTGTTAGAACAGTACACGGCTGGTGTAAAGAGACACTGTCCTGAAACTCAGCCCACAACTAAGCCGCTGACTAATGATAACATGGTTTCTAAGGAGAATCATTGTGGCAGCACATCTGGACCATCTCGTCCTCGAAATCGCTTTGCAACCCTCTTGCAGTGGAGAAACCGATCAGAAGAGGGCACTGAAGAGCAGGGCACATGCAGCAGGTTCTTCTGTCATGATGAATCCAACATAGCAGAGACACAGGAAGACTCAAAACAAGACTCTTCACAGTCTGTGGAGTCTCAAGAGAAGCATGTGTCACAGTCTGGAGGAGACACATCATCACTGTGTGAAGACCCAGAGAGAGAACAGGACAAAGATGAGCCATCTAGCCCACCCGCATCTCCATCTTGCTCTTCCAGGCCTGCTAGTGTGGGTCTTGGGGTCTTCAGCTGGTCAGGGAGCACCAAAGAACTCAATAAATCTGTTTCACATCCAGCCAGAGACAGCACAGAGAGACAGCGGTCATCCTCCACACCTTCAGGATTATCTACACTGCAGCAGTTTCACAGAAATAAGGCAAGAATCTCCTGGGCAGGACCTGGGCTTTCTCTCTCGTCCTCACCTGTTGAAGGCAGTGAGGATGCTGGAAATTCTCCCGGGTCTCCTCCATCTCAGGACAGTGCCTATTTCTCCCAGTCCAGCAGTATCTCTGCTAGTGTGGAGAACTCTTTAGTCACAGAAGACAACTCTGATAAGGAAAAGGAGAGAGATTCAGACGTCTCCAACAGCCCTAGCAGCTCTCCACTAGACAGGCTGAAACCTGCTGTTAACAGAACAAAGGCAAGGTTGTCCCGGAAAGGGGCTTGTGGGCAGGGTAAAGGCGGTAAAATTGAGACCTCCGCTCCTGCTAGAGCCAGCGGTCTGAGAAGGAAACCAAGCGGGAAGAAAAATGTCAACAATGAGAACAGCCCAGGTCTGCAGGCCACCATCAGTGGCCTCTGGGGGGCCTTCAGCTTCAAGAAGGACAGTCCAAAGTTAAGTGCAACCAAGAAAGGAGAGCCGATGTCACCGGTTGGGGAGAATGTGGTGATGGAAACGACTCAAGCTGATAAAGAGATTTTCATCATTGCTGAATGACATGTCACTGTTTCAACTGAATAACTTTTTTAAGAGTATCAGACAGTCTTTCAGACATACTGTGTATGTTAATGATTTGTAAGATAAATCAAACTTTTGTAAAATTATGAATTTTATTTCAAGTCAATAAAGACGTTATTCTTTA

XPG

AAACACAAGTCCAATGTCTCTAAAACAATGTTAAGTTTTCTTTACGCAAGGCAAACTGGGCTTGACGACCCAGTTCGACTGAGACGTGCAGAATCCACACGCAGGGTGCTGAGTCTTGAGTTGAACCACAACAGGGATGTGGATCGTATTCATGGCAATGGAATCAACACCCTTGACATTGAGGTTATTGATGGAAGATATATGCTGTCAGGCGGATCTGATGGCGTAATTGTGATTTATGACTTAGAGAACAACAGCAAGAAGCCTCAATACACCTGCAAAGCAATCTGCACTGTGGGCAGTCGACATGTGCATAAATTCAGCGTGGAAACAGTGCAGTGGTACCCACATGACACCGGTATGTTTGTATCCAGCTCTTTCGACAAAACCATGAAAGTCTGGGATGCTGAAACTCTGAAGCCAGCTGATGAGTTCCAGTTTGATGGCAATGTCTACTGTCATCACATGTCTCCTATAGCCCGGAAGCACAGCCTTGTGGCAGTTGGTACCAAAGACCCCAAAGTGCAGCTTTGTGATCTGAAGTCTGGGTCGCGTATCCACATCCTTCAGGGTCACAGGGGCGAGATCCTCTCAGTGAAATGGTCTCCACGATATGAACACATACTGGCCACTGCCAGCACTGACAGTAGGGTTCGCATCTGGGATGTGAGAAGAGCCTCCGGGAGTCTTTTTACTTTGGACCAGCACAATGGAGATAAATCCAAGGCCTCGTCTGAAGCAGTGAACACAGCACACAACGGAAGAGTGAATGGGTTGTGTTTCACAGCTGATGGACTCCACCTCCTCACTACAGGGACAGATGATCGCATGCGTCTGTGGAATAGCGGCACTGGGGAAAACACATTAGTGAACTATGGGAAGGTCGTGAACGAGAGCCGCAAAGGTCTGAAGTTTACCGTATCAAGGGGTTGCAGTCCAGAGTTTGTGTTTGTGCCATGTGGAAGCTCAGTAGCGGTGTATGGGCTTCATTCTGGAGAGCTGATCACTATGTTAAGGGGACATTATAATAATGTGGACTGCTGCGAGTTCCACCCTGATTATCAGGAGCTGTACAGTGGAGGTAAAGACTGTAACATACTTGCCTGGGTTCCAGTGTTAAGACAGCCTGATGTTGATGATGAAGAAACCAACACGAAACGGGGTGGAACGCAAGCTGCTATCAATCCTGCTTTTGCCGATGCCTGGAGCAGCGACGAAGACTGACAGATCAACCCAGCAATGTTTTTTTATATCCAAATACACCACTTTATACATTATTAAAGAAGTCAAAGTGCTCTTATTGCATTCAGAGGCACAGCAGGTGCATATAAGCTCAATGTACAGAAAAAAAAGACTTGTGGGAGATTTGTTCTCATTGCTTGTGACCATCTGATTTGCATG

CSA

ATTATTAACATTTTCATTTAAGTTCTCTTTTGACTTAACGCTGATCTGGTAAGACAAATCTTCATGAACACCTCGCTAGCTGATCATGGAGATGCCAGAGGCCAGTCATTGTCAACACACATCGCCAGACTCTGCAAACTCTTCAAATGAGTCCAGCATGTCTTTAACACCACACATGCTGGCCAAAATTGAGCGAAACCGCCAACGGGCGCTGATGCTCAGACAAGCCAGACTCGCCAGCAGACCGTCCTCCGCTGTAGAAGGAGCTACTCACGCTAAAGTGGCAAAAACTATAGACTCTGGTGCTGGGTTTTTTATTGAAGAGGAGACAACAGAGGACGAGCAGCAGGAGAAGAGAGTCGTTCAACAACCAGCCCCAGTGATGGAGCCTGATTATCTGATGTGCGAAGAGTGTCAAAAGCCATTTATGGATTCTTATCTGAGCAACAGTTTTGATCTGTCTGTGTGTGATAAATGCAGAGACAATGAAGTAAAGCACAAGCTAATATCTCGCACGGAAGCGAAAAAGAATTACCTCCTGAAGGACTGTGATCTGGATAAGAGGGAGCCACCGCTGCGATTCATCCTGAGGAAAAATCCTCACAATCCACGCTGGGGAGACATGAAACTCTACTTAAAGACACAGGTTGAGAAACGCTCTATGGAGGTCTGGGGCAGCGATGAGGCTCTGGAAGAGGCCAAGGAGACTAGAGAGGAGAACAAAGAGGTGCAGAAGCAGAAACGCTTTAATAAAAAAGTCAAAGAGCTGAGGCGGACGGTGAGGAGCAGTATGTTTAAGAAAGACACCAGCATTCATCAGCACGACTATGGCCATGACGAGCTGCTGGATGAAGAGGAAGATTCTTTCAGTGAAAAGTGTAACTTCAGAGATCCTACCTGTGTAAATACAGCCAACATTAGCACTCAGAAGATGTTTTCTAGTAGAAACTACACCTTAAAAACGCACAAATCCCCTTTATCCTTGAGAATATGTCAGAAAGGTGAAGAAAAGCAACAGAAAGATCTGCAGCAGCCAGACTCTGACAATTATCTAACCATCCCCATTAAAGCTCATCCTTCAGATTCAAAACCCTCACATGCATCGGGCTGCACCAAGTCTCACCTAAATACCTCCCACCTTCAGCCCGTGCAGCGCTCGCCTGTCATAATGGAGTCTTGGTCCCCTGACAGCCCTACAGCTATGATTTATCATCACGCTGTGCCCGTGCCAAATGTACCTGTTGTTCAACCCCAGCTGCTCTGCCTCACGCCCCCTGCAGACGTCCCGCATGCTCACATCCAGCGCAAGCTGCTGCTTGACCCCACCACGGGTCAGTACTACTTAGTGGACACCCCTGTTCCGATGCAGCCCGCCGCCCAGAGGTTTTACCATCCTGAGAGTGGCCAGTACTTGGACATTCCTTTATCACCACTCCAGTGCCAGTGTCGGTGTCACCGGTCGCCCTCGGTCCTGCAGCCTATCCCCCAACTTACTTGGTCTACCCTTCGACCTTTCTGCCTGCACCCCCGGCGCCGCAGTCACATTCATCCACCTGCTCCGAGGGCGAGGATGCAGTGGAGACGGGCAGCATGTATATGATCCCGCCGGGCAGCACAGCACCCAGCAGCACCACCAAACCGGTCATCAGCATCACATCGCAGCAAGGTGCCCGTATCGTTGCTCCTCCCTCCTTTGATGGCACAACAATGAGCTTTGTGGTGGAGCATCGATAACGGAAGGAGGATCCCAGTCAGAGTGTGTTCACCAACCACTTCATCAAAAAAAAAAAAAAAAA

XPA

Repair Enzyme Homology Repair Enzyme Homology between different speciesbetween different species

CSA: 92%

XPA: 86%

XPG: 92%

CSA: 85%

XPA: 75%

XPG: 60%

CSA: 86%

XPA: 72%

XPG: 61%

CSA: 86%

XPA: 64%, 78%

XPG: 60%

CSA: 84%

XPA: 73%

XPG: 48%

Fugu X. laevis and X. tropicalis

H. sapiens R. norvegicus M. musculus

XPA Phylogenetic XPA Phylogenetic AnalysisAnalysis

XPA Phylogenetic XPA Phylogenetic AnalysisAnalysis

CSA Phylogenetic CSA Phylogenetic AnalysisAnalysis

CSA Phylogenetic CSA Phylogenetic AnalysisAnalysis

Real Time PCRReal Time PCRTotal Zebrafish RNA

Oligo dT primersSuperscript RTRNase inhibitorprimer

primerFirst strand cDNA

Amplify cDNASPP

SPPPCR

Amplified product

RT-PCR and amplification of XPA, CSA and XPG cDNA

SPP: Specific pair primer for each gene

Real Time PCRReal Time PCR

1: STD control

2: 12 hr

3: 24 hr

4: 36 hr

5: 48 hr

6: (-)RT control

200 bp

CSA XPA XPG

1 2 3 4 5 6

1 2 3 4 5 6

1 2 3 4 5 6

wat

er

Experimental SchemeExperimental Scheme

embryo

Microinjection Translation inhibition

DNAProtein

Western Blot

DNA adduct analysis

Treat with DBP

DNA adduct analysis

DNA

embryo

Western Blot

Protein

Treat with DBP

MorpholinosMorpholinos

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 ~~

tRNA

40S

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 ~~

Protein

60S

40S

Protein

MorpholinosMorpholinos Inject the designed morpholino into the Inject the designed morpholino into the

developing embryo at the 1-cell stage.developing embryo at the 1-cell stage. Once incorporated into the embryo, the Once incorporated into the embryo, the

morpholino blocks translation of the gene.morpholino blocks translation of the gene.

A

O

O

O

O

O

OH

NH

OH OO

Morpholino

3’-Carboxyfluorescein

48 hpf

Future WorkFuture Work Continue with morpholino injections Continue with morpholino injections

and establish that the genes are and establish that the genes are knocked down.knocked down.

Isolate DNA from injected embryos Isolate DNA from injected embryos and look for any change in amount and look for any change in amount of DNA damage caused by DBP. of DNA damage caused by DBP.

AcknowledgementsAcknowledgementsHoward Hughes Medical Howard Hughes Medical

InstituteInstituteKevin AhernKevin Ahern

William BairdWilliam BairdRobert TanguayRobert Tanguay

Brinda MahadevanBrinda MahadevanEric AndreasenEric AndreasenMark Reimers Mark Reimers

Jane LaDuJane LaDuLijoy MathewLijoy MathewEric BrooksEric Brooks