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Role of DNA repair enzymes in removal of bulky DNA adducts in zebrafish

Role of DNA repair enzymes in removal of bulky DNA adducts in zebrafish. Jennifer Atkin Dr. William Baird. Smog!. Cigarette smoking!. Significance. CH 2 OH. O. HO. N. N. N. N. NH. HO. HO. OH. Adenosine (blue) adduct formed by reaction of Dibenzo[ a,I ]pyrene (red) with DNA .

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Role of DNA repair enzymes in removal of bulky DNA adducts in zebrafish

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  1. Role of DNA repair enzymes in removal of bulky DNA adducts in zebrafish Jennifer Atkin Dr. William Baird

  2. Smog! Cigarette smoking! Significance

  3. CH2OH O HO N N N N NH HO HO OH Adenosine (blue) adduct formed by reaction of Dibenzo[a,I]pyrene (red) withDNA DNA Adduct Formation • Polycyclic Aromatic Hydrocarbons (PAH) such as DBP are environmental carcinogens found in cigarette smoke, diesel exhaust, and other products of combustion.

  4. XPA XPG XPG CSA DNA Repair = DNA adduct

  5. 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).

  6. Sequencing Identifying and cloning genes Search for the genes on the web (www.ensembl.org) Design primers and amplify the genes via PCR

  7. XPG GTAACTTTACTTGGTGGTAGAAGCGCGAGCAAAAGCTCTCGGGTGACAGGCTGAACTCTTTTAAACCATCTTCCTCAGACATCTGTCCTTCCAGACAGACAACATGGGTATTCAGGGCCTCTTGCAGTTTATCAAAGATGCTTCGGAGCCCATGCATGTGAAGAAGTATCGAGGACAGACAGTGGCTGTGGACACATACTGCTGGCTTCATAAAGCAGCTTTTTCATGTGCAGAGAAGCTTGCAAAAGGGGAACCTACAGATCAGTATGTCTCCTACTGTATGAAGTTTGTGGACATGCTGCTTTCTTTTGGTGTTAAACCTATCTTGGTGTTTGATGGTCGTAACTTGCCCTCCAAACAGGAAGTGGAGAAGTCCCGGCGAGAGAGGCGACAGGCCAATCTGCAGAAAGGCAAACAGCTGCTGCGGGAGGGAAAGATAACAGAAGCCAGAGAATGTTTTACCCGAAGTGTTAACATTACCCCATCTATGGCACATGATGTCATTAGGGCTGCCAGAACACGTGGAGTGGACTGTGTTGTTGCTCCATATGAAGCAGATGCACAGTTAGCTTTCCTTAACAAATCTGATATCGCTCAGGCGGTGATCACAGAGGACTCAGATCTGCTGGCATTTGGCTGCAAAAAGGTCATTTTAAAGATGGATAAACAGGGTAATGGATTGGAGATTGAGCAGTGTCACCTGGGCCGATGCAAATCTCTTGGCAACATCTTCACAGAGGAGAAGTTTCGATATATGTGCATCCTCTCTGGCTGTGACTACTTGCAGTCTCTGTATGGTATTGGTCTGGGAAAAGCATGCAAGCTGCTCAGGATGGCAAATAACCCTGATATCCTGAAGGTGATCAAGAAGATGGGGCAGTATTTGAAGATGGACATTTCAGTTCCAGAAGAATACATTGAGGGTTTCACAAAAGCCAACAACACATTTTTGTATCAGCTGGTCTTTGACCCCTTGAGGAGAAAGGTGGTACCTTTGAACCCTTATCCAGATCACATTAATCCTGCTGCCCTCAGCTATGCTGGAACTAACGTAGGTGATGAGAAAGGGCTGCAAATGGCTCTTGGGAACCTGGACATTAACACAATGCAAAGAATAGATGACTTTAACCCTGACGCTCCTCAAACTCAGCCACCTAAAGCTCCTCGCAGTAGTTCCTGGAATGACAGATGTGACAAAACTGCCACAACACAGGCAAGCATTTGGAGCCAGAACTATGAACCAGGTTGCACCAAGCCACAGTCTCCTACCTCCCCTAAAAGACCACCACCCACCCGGGGCAAAGAGAGGATTGTTAGTGTGCAGAGTCTAAAGTTGCCCCAGAGAGAGTCACAGGTGAAAAGACCCCGTGAAGACACAAGTCTCTCTGTGGATGACCTGTTAGAACAGTACACGGCTGGTGTAAAGAGACACTGTCCTGAAACTCAGCCCACAACTAAGCCGCTGACTAATGATAACATGGTTTCTAAGGAGAATCATTGTGGCAGCACATCTGGACCATCTCGTCCTCGAAATCGCTTTGCAACCCTCTTGCAGTGGAGAAACCGATCAGAAGAGGGCACTGAAGAGCAGGGCACATGCAGCAGGTTCTTCTGTCATGATGAATCCAACATAGCAGAGACACAGGAAGACTCAAAACAAGACTCTTCACAGTCTGTGGAGTCTCAAGAGAAGCATGTGTCACAGTCTGGAGGAGACACATCATCACTGTGTGAAGACCCAGAGAGAGAACAGGACAAAGATGAGCCATCTAGCCCACCCGCATCTCCATCTTGCTCTTCCAGGCCTGCTAGTGTGGGTCTTGGGGTCTTCAGCTGGTCAGGGAGCACCAAAGAACTCAATAAATCTGTTTCACATCCAGCCAGAGACAGCACAGAGAGACAGCGGTCATCCTCCACACCTTCAGGATTATCTACACTGCAGCAGTTTCACAGAAATAAGGCAAGAATCTCCTGGGCAGGACCTGGGCTTTCTCTCTCGTCCTCACCTGTTGAAGGCAGTGAGGATGCTGGAAATTCTCCCGGGTCTCCTCCATCTCAGGACAGTGCCTATTTCTCCCAGTCCAGCAGTATCTCTGCTAGTGTGGAGAACTCTTTAGTCACAGAAGACAACTCTGATAAGGAAAAGGAGAGAGATTCAGACGTCTCCAACAGCCCTAGCAGCTCTCCACTAGACAGGCTGAAACCTGCTGTTAACAGAACAAAGGCAAGGTTGTCCCGGAAAGGGGCTTGTGGGCAGGGTAAAGGCGGTAAAATTGAGACCTCCGCTCCTGCTAGAGCCAGCGGTCTGAGAAGGAAACCAAGCGGGAAGAAAAATGTCAACAATGAGAACAGCCCAGGTCTGCAGGCCACCATCAGTGGCCTCTGGGGGGCCTTCAGCTTCAAGAAGGACAGTCCAAAGTTAAGTGCAACCAAGAAAGGAGAGCCGATGTCACCGGTTGGGGAGAATGTGGTGATGGAAACGACTCAAGCTGATAAAGAGATTTTCATCATTGCTGAATGACATGTCACTGTTTCAACTGAATAACTTTTTTAAGAGTATCAGACAGTCTTTCAGACATACTGTGTATGTTAATGATTTGTAAGATAAATCAAACTTTTGTAAAATTATGAATTTTATTTCAAGTCAATAAAGACGTTATTCTTTA XPA ATTATTAACATTTTCATTTAAGTTCTCTTTTGACTTAACGCTGATCTGGTAAGACAAATCTTCATGAACACCTCGCTAGCTGATCATGGAGATGCCAGAGGCCAGTCATTGTCAACACACATCGCCAGACTCTGCAAACTCTTCAAATGAGTCCAGCATGTCTTTAACACCACACATGCTGGCCAAAATTGAGCGAAACCGCCAACGGGCGCTGATGCTCAGACAAGCCAGACTCGCCAGCAGACCGTCCTCCGCTGTAGAAGGAGCTACTCACGCTAAAGTGGCAAAAACTATAGACTCTGGTGCTGGGTTTTTTATTGAAGAGGAGACAACAGAGGACGAGCAGCAGGAGAAGAGAGTCGTTCAACAACCAGCCCCAGTGATGGAGCCTGATTATCTGATGTGCGAAGAGTGTCAAAAGCCATTTATGGATTCTTATCTGAGCAACAGTTTTGATCTGTCTGTGTGTGATAAATGCAGAGACAATGAAGTAAAGCACAAGCTAATATCTCGCACGGAAGCGAAAAAGAATTACCTCCTGAAGGACTGTGATCTGGATAAGAGGGAGCCACCGCTGCGATTCATCCTGAGGAAAAATCCTCACAATCCACGCTGGGGAGACATGAAACTCTACTTAAAGACACAGGTTGAGAAACGCTCTATGGAGGTCTGGGGCAGCGATGAGGCTCTGGAAGAGGCCAAGGAGACTAGAGAGGAGAACAAAGAGGTGCAGAAGCAGAAACGCTTTAATAAAAAAGTCAAAGAGCTGAGGCGGACGGTGAGGAGCAGTATGTTTAAGAAAGACACCAGCATTCATCAGCACGACTATGGCCATGACGAGCTGCTGGATGAAGAGGAAGATTCTTTCAGTGAAAAGTGTAACTTCAGAGATCCTACCTGTGTAAATACAGCCAACATTAGCACTCAGAAGATGTTTTCTAGTAGAAACTACACCTTAAAAACGCACAAATCCCCTTTATCCTTGAGAATATGTCAGAAAGGTGAAGAAAAGCAACAGAAAGATCTGCAGCAGCCAGACTCTGACAATTATCTAACCATCCCCATTAAAGCTCATCCTTCAGATTCAAAACCCTCACATGCATCGGGCTGCACCAAGTCTCACCTAAATACCTCCCACCTTCAGCCCGTGCAGCGCTCGCCTGTCATAATGGAGTCTTGGTCCCCTGACAGCCCTACAGCTATGATTTATCATCACGCTGTGCCCGTGCCAAATGTACCTGTTGTTCAACCCCAGCTGCTCTGCCTCACGCCCCCTGCAGACGTCCCGCATGCTCACATCCAGCGCAAGCTGCTGCTTGACCCCACCACGGGTCAGTACTACTTAGTGGACACCCCTGTTCCGATGCAGCCCGCCGCCCAGAGGTTTTACCATCCTGAGAGTGGCCAGTACTTGGACATTCCTTTATCACCACTCCAGTGCCAGTGTCGGTGTCACCGGTCGCCCTCGGTCCTGCAGCCTATCCCCCAACTTACTTGGTCTACCCTTCGACCTTTCTGCCTGCACCCCCGGCGCCGCAGTCACATTCATCCACCTGCTCCGAGGGCGAGGATGCAGTGGAGACGGGCAGCATGTATATGATCCCGCCGGGCAGCACAGCACCCAGCAGCACCACCAAACCGGTCATCAGCATCACATCGCAGCAAGGTGCCCGTATCGTTGCTCCTCCCTCCTTTGATGGCACAACAATGAGCTTTGTGGTGGAGCATCGATAACGGAAGGAGGATCCCAGTCAGAGTGTGTTCACCAACCACTTCATCAAAAAAAAAAAAAAAAA CSA AAACACAAGTCCAATGTCTCTAAAACAATGTTAAGTTTTCTTTACGCAAGGCAAACTGGGCTTGACGACCCAGTTCGACTGAGACGTGCAGAATCCACACGCAGGGTGCTGAGTCTTGAGTTGAACCACAACAGGGATGTGGATCGTATTCATGGCAATGGAATCAACACCCTTGACATTGAGGTTATTGATGGAAGATATATGCTGTCAGGCGGATCTGATGGCGTAATTGTGATTTATGACTTAGAGAACAACAGCAAGAAGCCTCAATACACCTGCAAAGCAATCTGCACTGTGGGCAGTCGACATGTGCATAAATTCAGCGTGGAAACAGTGCAGTGGTACCCACATGACACCGGTATGTTTGTATCCAGCTCTTTCGACAAAACCATGAAAGTCTGGGATGCTGAAACTCTGAAGCCAGCTGATGAGTTCCAGTTTGATGGCAATGTCTACTGTCATCACATGTCTCCTATAGCCCGGAAGCACAGCCTTGTGGCAGTTGGTACCAAAGACCCCAAAGTGCAGCTTTGTGATCTGAAGTCTGGGTCGCGTATCCACATCCTTCAGGGTCACAGGGGCGAGATCCTCTCAGTGAAATGGTCTCCACGATATGAACACATACTGGCCACTGCCAGCACTGACAGTAGGGTTCGCATCTGGGATGTGAGAAGAGCCTCCGGGAGTCTTTTTACTTTGGACCAGCACAATGGAGATAAATCCAAGGCCTCGTCTGAAGCAGTGAACACAGCACACAACGGAAGAGTGAATGGGTTGTGTTTCACAGCTGATGGACTCCACCTCCTCACTACAGGGACAGATGATCGCATGCGTCTGTGGAATAGCGGCACTGGGGAAAACACATTAGTGAACTATGGGAAGGTCGTGAACGAGAGCCGCAAAGGTCTGAAGTTTACCGTATCAAGGGGTTGCAGTCCAGAGTTTGTGTTTGTGCCATGTGGAAGCTCAGTAGCGGTGTATGGGCTTCATTCTGGAGAGCTGATCACTATGTTAAGGGGACATTATAATAATGTGGACTGCTGCGAGTTCCACCCTGATTATCAGGAGCTGTACAGTGGAGGTAAAGACTGTAACATACTTGCCTGGGTTCCAGTGTTAAGACAGCCTGATGTTGATGATGAAGAAACCAACACGAAACGGGGTGGAACGCAAGCTGCTATCAATCCTGCTTTTGCCGATGCCTGGAGCAGCGACGAAGACTGACAGATCAACCCAGCAATGTTTTTTTATATCCAAATACACCACTTTATACATTATTAAAGAAGTCAAAGTGCTCTTATTGCATTCAGAGGCACAGCAGGTGCATATAAGCTCAATGTACAGAAAAAAAAGACTTGTGGGAGATTTGTTCTCATTGCTTGTGACCATCTGATTTGCATG Zebrafish Repair Gene Sequences

  8. CSA: 92% XPA: 86% XPG: 92% CSA: 86% XPA: 64%, 78% XPG: 60% CSA: 86% XPA: 72% XPG: 61% CSA: 84% XPA: 73% XPG: 48% CSA: 85% XPA: 75% XPG: 60% Repair Enzyme Homology between different species Fugu X. laevis and X. tropicalis R. norvegicus M. musculus H. sapiens

  9. XPA Phylogenetic Analysis

  10. XPA Phylogenetic Analysis

  11. CSA Phylogenetic Analysis

  12. CSA Phylogenetic Analysis

  13. Real Time PCR RT-PCR and amplification of XPA, CSA and XPG cDNA Total Zebrafish RNA Oligo dT primers Superscript RT RNase inhibitor primer First strand cDNA primer SPP Amplify cDNA SPP PCR Amplified product SPP: Specific pair primer for each gene

  14. CSA XPA XPG water 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 200 bp Real Time PCR 1: STD control 2: 12 hr 3: 24 hr 4: 36 hr 5: 48 hr 6: (-)RT control

  15. embryo Microinjection Translation inhibition Treat with DBP embryo Protein DNA Protein DNA Western Blot DNA adduct analysis Western Blot DNA adduct analysis Experimental Scheme Treat with DBP

  16. G T T G T A C C C A T A A G T C C C G G A G A A Protein 60S 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 ~~ ~~ 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 40S Protein tRNA Morpholinos

  17. Morpholino 3’-Carboxyfluorescein 48 hpf 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.

  18. 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.

  19. Howard Hughes Medical Institute Kevin Ahern William Baird Robert Tanguay Brinda Mahadevan Eric Andreasen Mark Reimers Jane LaDu Lijoy Mathew Eric Brooks Acknowledgements

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