1. Presenter: Cassandra Lanette Carr, Claflin University Mentor, Dr. M. Wyatt, Coker Life Sciences (USC) The Cancer Research Training Program November 16, 2004 DNA REPAIR Quality Control by DNA Repair

2. DNA Double Helix

3. DNA Base Pairs AcceptorsHydrogen donorsGlycosidic bonds CH 3 Major Groove Minor Groove Major Groove Minor Groove

4. DNA base modifications can be toxic or mutagenic 3-Methyladenine is toxic because it blocks DNA polymerases Hypoxanthine (Hx, deaminated adenine) is mutagenic because DNA polymerases mis-insert cytosine Background Information

5. What causes DNA damage? Replication errors (base:base mismatches, insertion/deletion loops) Oxidative/hydrolytic damage (base damage, base loss) UV and x-rays Carcinogens (alkylation damage) –nitrosoamines, benzo[a]pyrene, aflatoxin Most cancer chemotherapeutic drugs

6. DNA Repair Systems From Science, 1999, p. 1897

7. OH PCNA polymerase  FEN1 DNA ligase Short patch (major)Long patch (minor) BER Pathway DNA glycosylase AP endonuclease OH polymerase  DNA ligase

8. Nature 411,366-74

9. Substrates include: –incorrect bases (e.g., uracil in DNA) –deaminated and oxidized bases –alkylated bases DNA glycosylases remove incorrect or damaged bases

10. DNA glycosylase DNA Glycosylase

11. Human 3-methyladenine DNA Glycosylase (AAG) Wide substrate range, removing a variety of damaged bases 3-methyladenine DNA glycosylases protect cells from methyl methanesulfonate (MMS) toxicity adenine methylation 3-methyladenine

12. AAG Substrates (7-MeG)(3-MeA)

13. Data

14. Glucose Plates Control 0.015% of MMS 0.025% of MMS Results MMS Gradient Plates Wild type E125Q Y165A L180S Low concentration High concentration

15. Galactose Plates Control 0.025% of MMS 0.03% of MMS More MMS Gradient Plates Low concentrationHigh concentration Wild type E125Q Y165A L180S

16. Chloroacetylaldehyde (CAA) introduces etheno-base damage. AAG Substrates

17. CCAA Gradient Plates Glucose Plates Control 0.003% of CAA 0.006% of CAA Wild type E125Q N169D N169S Low concentrationHigh concentration

18. CAA Gradient Plates Galactose Plates Control 0.003% of CAA 0.006% of CAA Wild type Low concentrationHigh concentration E125Q N169D N169S

19. The purpose of the gradient plate assay To qualitatively assess glycosylase activity by measuring the survival of yeast when challenged with DNA damaging agents MMS and CAA What is different about CAA versus MMS MMS creates methylation damage 3-methyladenine is very toxic –Wild-type 3-methyladenine DNA glycosylase protects the yeast from MMS toxicity CAA creates etheno-base damage etheno-adenine is toxic and very mutagenic –Wild-type 3-methyladenine DNA glycosylase protects the yeast from CAA toxicity and mutagenicity Conclusion

20. Future Research Finish more plates using CAA to gather further results Start working on a plasmid miniprep kit ­This kit is designed to extract a DNA plasmid from a host cell